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The DNA-binding protein PfAP2-G is found to be a master regulator of sexual development in the malaria parasite; this protein appears to regulate early gametocytogenesis and is epigenetically silenced in the majority of blood-stage parasites. Malarial virulence factor primed for action For malaria parasites to be transmitted to the mosquito vector they must undergo sexual development and produce gametocytes. The molecular mechanisms underlying the commitment to gametocyte development have been unclear. Two complementary manuscripts now show that AP2-G, a member of the apicomplexan AP2 family of transcription factors, is a master regulator of sexual development in the malaria parasite, acting as a developmental switch by triggering the transcription of early gametocyte genes. Abhinav Sinha et al . worked with the rodent malaria parasite Plasmodium berghei , and Björn Kafsack et al . with the human pathogen P. falciparum . AP2-G activity in human infectious malaria parasites could be a potential target for antimalarials designed to interfere with gametocyte formation. The life cycles of many parasites involve transitions between disparate host species, requiring these parasites to go through multiple developmental stages adapted to each of these specialized niches. Transmission of malaria parasites ( Plasmodium spp.) from humans to the mosquito vector requires differentiation from asexual stages replicating within red blood cells into non-dividing male and female gametocytes. Although gametocytes were first described in 1880, our understanding of the molecular mechanisms involved in commitment to gametocyte formation is extremely limited, and disrupting this critical developmental transition remains a long-standing goal 1 . Here we show that expression levels of the DNA-binding protein PfAP2-G correlate strongly with levels of gametocyte formation. Using independent forward and reverse genetics approaches, we demonstrate that PfAP2-G function is essential for parasite sexual differentiation. By combining genome-wide PfAP2-G cognate motif occurrence with global transcriptional changes resulting from PfAP2-G ablation, we identify early gametocyte genes as probable targets of PfAP2-G and show that their regulation by PfAP2-G is critical for their wild-type level expression. In the asexual blood-stage parasites pfap2-g appears to be among a set of epigenetically silenced loci 2 , 3 prone to spontaneous activation 4 . Stochastic activation presents a simple mechanism for a low baseline of gametocyte production. Overall, these findings identify PfAP2-G as a master regulator of sexual-stage development in malaria parasites and mark the first discovery of a transcriptional switch controlling a differentiation decision in protozoan parasites.

Fanconi Anemia (FA) is a genomic instability syndrome resulting in aplastic anemia, developmental abnormalities, and predisposition to hematological and other solid organ malignancies. Mutations in genes that encode proteins of the FA pathway fail to orchestrate the repair of DNA damage caused by DNA interstrand crosslinks. Zebrafish harbor homologs for nearly all known FA genes. We used multiplexed CRISPR/Cas9-mediated mutagenesis to generate loss-of-function mutants for 17 FA genes: fanca, fancb, fancc, fancd1/brca2, fancd2, fance, fancf, fancg, fanci, fancj/brip1, fancl, fancm, fancn/palb2, fanco/rad51c, fancp/slx4, fancq/ercc4, fanct/ube2t, and two genes encoding FA-associated proteins: faap100 and faap24. We selected two indel mutations predicted to cause premature truncations for all but two of the genes, and a total of 36 mutant lines were generated for 19 genes. Generating two independent mutant lines for each gene was important to validate their phenotypic consequences. RT-PCR from homozygous mutant fish confirmed the presence of transcripts with indels in all genes. Interestingly, 4 of the indel mutations led to aberrant splicing, which may produce a different protein than predicted from the genomic sequence. Analysis of RNA is thus critical in proper evaluation of the consequences of the mutations introduced in zebrafish genome. We used fluorescent reporter assay, and western blots to confirm loss-of-function for several mutants. Additionally, we developed a DEB treatment assay by evaluating morphological changes in embryos and confirmed that homozygous mutants from all the FA genes that could be tested (11/17), displayed hypersensitivity and thus were indeed null alleles. Our multiplexing strategy helped us to evaluate 11 multiple gene knockout combinations without additional breeding. Homozygous zebrafish for all 19 single and 11 multi-gene knockouts were adult viable, indicating FA genes in zebrafish are generally not essential for early development. None of the mutant fish displayed gross developmental abnormalities except for fancp-/- fish, which were significantly smaller in length than their wildtype clutch mates. Complete female-to-male sex reversal was observed in knockouts for 12/17 FA genes, while partial sex reversal was seen for the other five gene knockouts. All adult females were fertile, and among the adult males, all were fertile except for the fancd1 mutants and one of the fancj mutants. We report here generation and characterization of zebrafish knockout mutants for 17 FA disease-causing genes, providing an integral resource for understanding the pathophysiology associated with the disrupted FA pathway.

The infraorder Brachyura (true or short-tailed crabs) represents a successful group of marine invertebrates yet with limited genomic resources. Here we report a chromosome-anchored reference genome and transcriptomes of the Chinese mitten crab Eriocheir sinensis , a catadromous crab and invasive species with wide environmental tolerance, strong osmoregulatory capacity and high fertility. We show the expansion of specific gene families in the crab, including F-ATPase, which enhances our knowledge on the adaptive plasticity of this successful invasive species. Our analysis of spatio-temporal transcriptomes and the genome of E. sinensis and other decapods shows that brachyurization development is associated with down-regulation of Hox genes at the megalopa stage when tail shortening occurs. A better understanding of the molecular mechanism regulating sexual development is achieved by integrated analysis of multiple omics. These genomic resources significantly expand the gene repertoire of Brachyura, and provide insights into the biology of this group, and Crustacea in general. Brachyurans, or crabs, are of commercial and ecological importance, but limited genomic resources exist. Here the authors present a chromosome-level genome and expression data for the Chinese mitten crab, to shed light on the biology of this group.

Malaria parasites must produce gametocytes for transmission to the mosquito vector, although the molecular mechanisms underlying commitment to gametocyte production remain unclear; here this process is found to be controlled by PbAP2-G, a member of the ApiAP2 family of DNA-binding proteins, in the rodent-infecting Plasmodium berghei parasite. Malarial virulence factor primed for action For malaria parasites to be transmitted to the mosquito vector they must undergo sexual development and produce gametocytes. The molecular mechanisms underlying the commitment to gametocyte development have been unclear. Two complementary manuscripts now show that AP2-G, a member of the apicomplexan AP2 family of transcription factors, is a master regulator of sexual development in the malaria parasite, acting as a developmental switch by triggering the transcription of early gametocyte genes. Abhinav Sinha et al . worked with the rodent malaria parasite Plasmodium berghei , and Björn Kafsack et al . with the human pathogen P. falciparum . AP2-G activity in human infectious malaria parasites could be a potential target for antimalarials designed to interfere with gametocyte formation. Commitment to and completion of sexual development are essential for malaria parasites (protists of the genus Plasmodium ) to be transmitted through mosquitoes 1 . The molecular mechanism(s) responsible for commitment have been hitherto unknown. Here we show that PbAP2-G, a conserved member of the apicomplexan AP2 (ApiAP2) family of DNA-binding proteins, is essential for the commitment of asexually replicating forms to sexual development in Plasmodium berghei , a malaria parasite of rodents. PbAP2-G was identified from mutations in its encoding gene, PBANKA_143750, which account for the loss of sexual development frequently observed in parasites transmitted artificially by blood passage. Systematic gene deletion of conserved ApiAP2 genes in Plasmodium confirmed the role of PbAP2-G and revealed a second ApiAP2 member (PBANKA_103430, here termed PbAP2-G2) that significantly modulates but does not abolish gametocytogenesis, indicating that a cascade of ApiAP2 proteins are involved in commitment to the production and maturation of gametocytes. The data suggest a mechanism of commitment to gametocytogenesis in Plasmodium consistent with a positive feedback loop involving PbAP2-G that could be exploited to prevent the transmission of this pernicious parasite.

Background Exploring the physiological and molecular mechanisms underlying goat sexual maturation can enhance breeding practices and optimize reproductive efficiency and is therefore substantially important for practical breeding purposes. As an essential neuroendocrine organ in animals, the hypothalamus is involved in sexual development and other reproductive processes in female animals. Although microRNAs (miRNAs) have been identified as significant regulators of goat reproduction, there is a lack of research on the molecular regulatory mechanisms of hypothalamic miRNAs that are involved in the sexual development of goats. Therefore, we examined the dynamic changes in serum hormone profiles and hypothalamic miRNA expression profiles at four developmental stages (1 day (neonatal, D1, n  = 5), 2 months (prepubertal, M2, n  = 5), 4 months (sexual maturity, M4, n  = 5), and 6 months (breeding period, M6, n  = 5)) during sexual development in Jining grey goats. Results Transcriptome analysis revealed 95 differentially expressed miRNAs (DEMs) in the hypothalamus of goats across the four developmental stages. The target genes of these miRNAs were significantly enriched in the GnRH signalling pathway, the PI3K-Akt signalling pathway, and the Ras signalling pathway ( P  < 0.05). Additionally, 16 DEMs are common among the M2 vs. D1, M4 vs. D1, and M6 vs. D1 comparisons, indicating that the transition from D1 to M2 represents a potentially critical period for sexual development in Jining grey goats. The bioinformatics analysis results indicate that miR-193a/miR-193b-3p-Annexin A7 ( ANXA7 ), miR-324-5p-Adhesion G protein-coupled receptor A1 ( ADGRA1 ), miR-324-3p-Erbb2 receptor tyrosine kinase 2 ( ERBB2 ), and miR-324-3p-Rap guanine nucleotide exchange factor 3 ( RAPGEF3 ) are potentially involved in biological processes such as hormone secretion, energy metabolism, and signal transduction. In addition, we further confirmed that miR-324-3p targets the regulatory gene RAPGEF3 . Conclusion These results further enrich the expression profile of hypothalamic miRNAs in goats and provide important insights for studying the regulatory effects of hypothalamic miRNAs on the sexual development of goats after birth.

Many metazoans switch between asexual and sexual reproduction based on environmental changes, life cycle phases, or both. This reproductive strategy enables them to benefit from the features of both reproductive modes. In general, asexual reproduction is broadly divided into parthenogenesis and vegetative reproduction. As in parthenogenesis, individuals develop ovaries and lay eggs, the most significant event in switching from parthenogenesis to sexual reproduction is the production of testes. Meanwhile, in vegetative reproduction, individuals do not need germ cells themselves. Thus, they must post-embryonically develop and maintain germ cells derived from pluripotent cells as they switch from vegetative to sexual reproduction. The complicated mechanisms for controlling the postembryonic reproductive development remain unknown. The planarian Dugesia ryukyuensis can switch from vegetative to sexual reproduction by stimulating bioactive compounds called sex-inducing substances, which are widely conserved in Platyhelminthes, including parasitic flatworms. The two reproductive modes are facilitated by the presence of adult pluripotent stem cells, which generate any type of somatic tissue in the asexual state and produce and maintain hermaphroditic reproductive organs in the sexual state. In this study, using RNA sequencing analysis in experimental sexualization by sex-inducing substances, we identified four essential genes for sexualization. A common feature following the knockdown of the four essential genes was a blockage of testicular differentiation. One of the four essential genes was a gap junction gene, Dr-siri ( D ugesia r yukyuensis - s exual i nduction- r elated i nnexin). We suggest that the establishment of a testicular stem cell niche supported by Dr-siri protein is responsible for the breakthrough of dormancy in postembryonic reproductive development in planarian reproductive switching. Our findings suggest that the production of testes might be crucial for even switching from vegetative to sexual reproduction.

The sexual Fus3 MAP kinase module of yeast is highly conserved in eukaryotes and transmits external signals from the plasma membrane to the nucleus. We show here that the module of the filamentous fungus Aspergillus nidulans (An) consists of the AnFus3 MAP kinase, the upstream kinases AnSte7 and AnSte11, and the AnSte50 adaptor. The fungal MAPK module controls the coordination of fungal development and secondary metabolite production. It lacks the membrane docking yeast Ste5 scaffold homolog; but, similar to yeast, the entire MAPK module's proteins interact with each other at the plasma membrane. AnFus3 is the only subunit with the potential to enter the nucleus from the nuclear envelope. AnFus3 interacts with the conserved nuclear transcription factor AnSte12 to initiate sexual development and phosphorylates VeA, which is a major regulatory protein required for sexual development and coordinated secondary metabolite production. Our data suggest that not only Fus3, but even the entire MAPK module complex of four physically interacting proteins, can migrate from plasma membrane to nuclear envelope.

Background The hypothalamus is a critical organ that regulates sexual development in animals. However, current research on the hypothalamic regulation of sexual maturation in female goats remains limited. In this study, we conducted metabolomic and transcriptomic analyses on the hypothalamic tissues of female Jining grey goats at different stages of sexual development (1 day old (neonatal, D1, n  = 5), 2 months old (prepuberty, M2, n  = 5), 4 months old (sexual maturity, M4, n  = 5), and 6 months old (breeding period, M6, n  = 5)). Results A total of 418 differential metabolites (DAMs) were identified in this study, among which the abundance of metabolites such as anserine, L-histidine, carnosine, taurine, and 4-aminobutyric gradually increased with the progression of sexual development. These metabolites may regulate neuronal development and hormone secretion processes by influencing the metabolism of histidine and phenylalanine. Through combined transcriptomic and metabolomic analyses, we identified that differentially expressed genes such as mitogen-activated protein kinase kinase kinase 9 ( MAP3K9 ), prune homolog 2 with BCH domain ( PRUNE2 ), and potassium voltage-gated channel interacting protein 4( KCNIP4 ) may jointly regulate the development and energy metabolism of hypothalamic Gonadotropin-releasing hormone neurons in conjunction with DAMs, including LPC22:5, 2-Arachidonyl Glycerol ether, LPE22:5, and Lysops22:5. Additionally, we elucidated the molecular mechanism through which glutathione metabolism regulates sexual maturation in goats. Conclusions In summary, this study illustrates the dynamic changes in metabolites and mRNA within hypothalamic tissue during postnatal sexual maturation in female Jining grey goats. This research may provide significant scientific insights for future animal breeding.

Seeds of flowering plants can be formed sexually or asexually through apomixis. Apomixis occurs in about 400 species and is of great interest for agriculture as it produces clonal offspring. It differs from sexual reproduction in three major aspects: (1) While the sexual megaspore mother cell (MMC) undergoes meiosis, the apomictic initial cell (AIC) omits or aborts meiosis (apomeiosis); (2) the unreduced egg cell of apomicts forms an embryo without fertilization (parthenogenesis); and (3) the formation of functional endosperm requires specific developmental adaptations. Currently, our knowledge about the gene regulatory programs underlying apomixis is scarce. We used the apomict Boechera gunnisoniana, a close relative of Arabidopsis thaliana, to investigate the transcriptional basis underlying apomeiosis and parthenogenesis. Here, we present the first comprehensive reference transcriptome for reproductive development in an apomict. To compare sexual and apomictic development at the cellular level, we used laser-assisted microdissection combined with microarray and RNA-Seq analyses. Conservation of enriched gene ontologies between the AIC and the MMC likely reflects functions of importance to germline initiation, illustrating the close developmental relationship of sexuality and apomixis. However, several regulatory pathways differ between sexual and apomictic germlines, including cell cycle control, hormonal pathways, epigenetic and transcriptional regulation. Enrichment of specific signal transduction pathways are a feature of the apomictic germline, as is spermidine metabolism, which is associated with somatic embryogenesis in various plants. Our study provides a comprehensive reference dataset for apomictic development and yields important new insights into the transcriptional basis underlying apomixis in relation to sexual reproduction.

The eukaryotic serine/threonine protein phosphatase PP2A is a heterotrimeric enzyme composed of a scaffold A subunit, a regulatory B subunit, and a catalytic C subunit. Of the four known B subunits, the B”’ subunit (known as striatin) interacts with the multi-protein striatin-interacting phosphatase and kinase (STRIPAK) complex. Orthologs of STRIPAK components were identified in Cryptococcus neoformans , namely PP2AA/Tpd3, PP2AC/Pph22, PP2AB/Far8, STRIP/Far11, SLMAP/Far9, and Mob3. Structural modeling, protein domain analysis, and detected protein-protein interactions suggest C . neoformans STRIPAK is assembled similarly to the human and fungal orthologs. Here, STRIPAK components Pph22, Far8, and Mob3 were functionally characterized. Whole-genome sequencing revealed that mutations in STRIPAK complex subunits lead to increased segmental and chromosomal aneuploidy, suggesting STRIPAK functions in maintaining genome stability. We demonstrate that PPH22 is a haploinsufficient gene: heterozygous PPH22/pph22 Δ mutant diploid strains exhibit defects in hyphal growth and sporulation and have a significant fitness disadvantage when grown in competition against a wild-type diploid. Deletion mutants pph22 Δ, far8 Δ, and mob3 Δ exhibit defects in mating and sexual differentiation, including impaired hyphae, basidia, and basidiospore production. Loss of either PPH22 or FAR8 in a haploid background leads to growth defects at 30°C, severely reduced growth at elevated temperature, abnormal cell morphology, and impaired virulence. Additionally, pph22 Δ strains frequently accumulate suppressor mutations that result in overexpression of another putative PP2A catalytic subunit, PPG1 . The pph22 Δ and far8 Δ mutants are also unable to grow in the presence of the calcineurin inhibitors cyclosporine A or FK506, and thus these mutations are synthetically lethal with loss of calcineurin activity. Conversely, mob3 Δ mutants display increased thermotolerance, capsule production, and melanization, and are hypervirulent in a murine infection model. Taken together, these findings reveal that the C . neoformans STRIPAK complex plays an important role in genome stability, vegetative growth, sexual development, and virulence in this prominent human fungal pathogen.