Stage-specific Gene Research Articles

Identification of two transcription factors that work coordinately to regulate early development in Entamoeba.

The protozoan parasite Entamoeba has a life cycle that switches between infective cysts and invasive trophozoites. Encystation, a crucial process in parasite biology, is controlled by different mechanisms including transcriptional control. We identified two nuclear proteins in Entamoeba invadens, EIN_066100 and EIN_085620, that regulate parasite development by binding to a DNA motif (TCACTTTC) in the promoter regions of genes upregulated in the first 8 h of stage conversion. Overexpression of EIN_066100, a homolog of MAK16 protein, resulted in reduced amoebic proliferation without affecting encystation efficiency. Overexpression of EIN_085620, a protein with an RNA-recognition motif (RRM), led to increased encystation efficiency. Glutathione S-transferase (GST) pull down assays revealed that EIN_066100 interacts with EIN_085620 both in vivo and in vitro, and this interaction is mediated by the EIN_085620 RRM domain. By evaluating truncated proteins with deletions at either the N-terminal or C-terminal regions of EIN_066100, we elucidated the importance of its N-terminal region in proper protein localization, proliferation, encystation, and interaction with EIN_085620. Taken together, these results indicate a coordinated role of EIN_066100 and EIN_085620 in regulating Entamoeba development. This work sheds light on the molecular mechanisms in the earliest stages of Entamoeba encystation.IMPORTANCEAn important biological process in the biology of Entamoeba is stage conversion, which plays a crucial role in disease propagation, facilitating parasite survival outside the host and spreading to new hosts. Multiple mechanisms contribute to controlling the expression of amebic stage-specific genes such as epigenetic and transcriptional control. Identification of early transcriptional control regulators is crucial to understanding the initiation of the encystation cascade. We identified two nuclear proteins, EIN_066100 and EIN_085620, involved in the proliferation and developmental regulation of E. invadens. These proteins work by direct binding to each other and mediating encystation efficiency. Study of new regulators involved in Entamoeba development represents an important advance in a critical aspect of parasite biology.

Emerging role of microglia in the developing dopaminergic system: perturbation by early life stress

Early life stress correlates with a higher prevalence of neurological disorders, including autism, attention-deficit/hyperactivity disorder, schizophrenia, depression, and Parkinson's disease. These conditions, primarily involving abnormal development and damage of the dopaminergic system, pose significant public health challenges. Microglia, as the primary immune cells in the brain, are crucial in regulating neuronal circuit development and survival. From the embryonic stage to adulthood, microglia exhibit stage-specific gene expression profiles, transcriptome characteristics, and functional phenotypes, enhancing the susceptibility to early life stress. However, the role of microglia in mediating dopaminergic system disorders under early life stress conditions remains poorly understood. This review presents an up-to-date overview of preclinical studies elucidating the impact of early life stress on microglia, leading to dopaminergic system disorders, along with the underlying mechanisms and therapeutic potential for neurodegenerative and neurodevelopmental conditions. Impaired microglial activity damages dopaminergic neurons by diminishing neurotrophic support (e.g., insulin-like growth factor-1) and hinders dopaminergic axon growth through defective phagocytosis and synaptic pruning. Furthermore, blunted microglial immunoreactivity suppresses striatal dopaminergic circuit development and reduces neuronal transmission. Furthermore, inflammation and oxidative stress induced by activated microglia can directly damage dopaminergic neurons, inhibiting dopamine synthesis, reuptake, and receptor activity. Enhanced microglial phagocytosis inhibits dopamine axon extension. These long-lasting effects of microglial perturbations may be driven by early life stress–induced epigenetic reprogramming of microglia. Indirectly, early life stress may influence microglial function through various pathways, such as astrocytic activation, the hypothalamic–pituitary–adrenal axis, the gut–brain axis, and maternal immune signaling. Finally, various therapeutic strategies and molecular mechanisms for targeting microglia to restore the dopaminergic system were summarized and discussed. These strategies include classical antidepressants and antipsychotics, antibiotics and anti-inflammatory agents, and herbal-derived medicine. Further investigations combining pharmacological interventions and genetic strategies are essential to elucidate the causal role of microglial phenotypic and functional perturbations in the dopaminergic system disrupted by early life stress.

Multi-organ gene expression analysis and network modeling reveal regulatory control cascades during the development of hypertension in female spontaneously hypertensive rat.

Hypertension is a multifactorial disease with stage-specific gene expression changes occurring in multiple organs over time. The temporal sequence and the extent of gene regulatory network changes occurring across organs during the development of hypertension remain unresolved. In this study, female spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats were used to analyze expression patterns of 96 genes spanning inflammatory, metabolic, sympathetic, fibrotic, and renin-angiotensin (RAS) pathways in five organs, at five time points from the onset to established hypertension. We analyzed this multi-dimensional dataset containing ~15,000 data points and developed a data-driven dynamic network model that accounts for gene regulatory influences within and across visceral organs and multiple brainstem autonomic control regions. We integrated the data from female SHR and WKY with published multiorgan gene expression data from male SHR and WKY. In female SHR, catecholaminergic processes in the adrenal gland showed the earliest gene expression changes prior to inflammation-related gene expression changes in the kidney and liver. Hypertension pathogenesis in male SHR instead manifested early as catecholaminergic gene expression changes in brainstem and kidney, followed by an upregulation of inflammation-related genes in liver. RAS-related gene expression from the kidney-liver-lung axis was downregulated and intra-adrenal RAS was upregulated in female SHR, whereas the opposite pattern of gene regulation was observed in male SHR. We identified disease-specific and sex-specific differences in regulatory interactions within and across organs. The inferred multi-organ network model suggests a diminished influence of central autonomic neural circuits over multi-organ gene expression changes in female SHR. Our results point to the gene regulatory influence of the adrenal gland on spleen in female SHR, as compared to brainstem influence on kidney in male SHR. Our integrated molecular profiling and network modeling identified a stage-specific, sex-dependent, multi-organ cascade of gene regulation during the development of hypertension.

An attractor state zone precedes neural crest fate in melanoma initiation.

The field cancerization theory suggests that a group of cells containing oncogenic mutations are predisposed to transformation 1, 2 . We previously identified single cells in BRAF V600E ;p53 -/- zebrafish that reactivate an embryonic neural crest state before initiating melanoma 3-5 . Here we show that single cells reactivate the neural crest fate from within large fields of adjacent abnormal melanocytes, which we term the "cancer precursor zone." These cancer precursor zone melanocytes have an aberrant morphology, dysplastic nuclei, and altered gene expression. Using single cell RNA-seq and ATAC-seq, we defined a distinct transcriptional cell attractor state for cancer precursor zones and validated the stage-specific gene expression initiation signatures in human melanoma. We identify the cancer precursor zone driver, ID1, which binds to TCF12 and inhibits downstream targets important for the maintenance of melanocyte morphology and cell cycle control. Examination of patient samples revealed precursor melanocytes expressing ID1, often surrounding invasive melanoma, indicating a role for ID1 in early melanomagenesis. This work reveals a surprising field effect of melanoma initiation in vivo in which tumors arise from within a zone of morphologically distinct, but clinically covert, precursors with altered transcriptional fate. Our studies identify novel targets that could improve early diagnosis and prevention of melanoma.

Inhibition of proteasome activity facilitates definitive endodermal specification of pluripotent stem cells by influencing YAP signalling

AimsThe knowledge of the molecular players that regulate the generation of endoderm cells is imperative to obtain homogenous population of pancreatic β-cells from stem cells. The Ubiquitin proteasome system (UPS) has been envisaged as a crucial intracellular protein degradation system, but its role in the generation of β-cells remains elusive. Hence, it would be appropriate to unravel the potential role of UPS in endoderm specification and utilize the understanding to generate β-cells from pluripotent stem cells. Materials and methodsThe pluripotent stem cells (mESCs, miPSCs and hIPSCs) were subjected to differentiation towards pancreatic β-cells and assessed the proteasomal activity during endodermal differentiation. Pharmacologic agents MG132 and IU-1 were employed to inhibit and activate proteasomal activity respectively at the definitive endoderm stage to investigate its impact on the generation of β-cells. The expression of stage-specific genes were analyzed at transcript and protein levels. We also explored the role of unfolded protein response and UPS-regulated signalling pathways in endodermal differentiation. Key findingsWe observed decreased proteasomal activity specifically during endoderm, but not during the generation of other lineages. Extraneous proteasomal inhibition enhanced the expression of endodermal genes while increasing the proteasomal activity hindered definitive endodermal differentiation. Proteasomal inhibition at the definitive endodermal stage culminated in an enriched generation of insulin-positive cells. Elevated endodermal gene expression was consistent in mESCs and hIPSCs upon proteasomal inhibition. Mechanistic insight revealed the proteasome-inhibited enhanced endodermal differentiation to be via modulating the YAP pathway. SignificanceOur study unravels the specific involvement of UPS in endoderm cell generation from pluripotent stem cells and paves the way for obtaining potential definitive endodermal cells for plausible cellular therapy in the future.

MRNA 5-methylcytosine in Eimeria tenella oocysts: An abundant post-transcriptional modification associated with broad-ranging biological processes

Eimeria tenella is the major causative agent of chicken coccidiosis. 5-Methylcytosine (m5C) is a type of RNA chemical modifications reported to regulate diverse biological processes. However, the distribution and biological functions of m5C in E. tenella mRNAs are yet to be known. Herein, we report transcriptome-wide profiling of mRNA m5C in E. tenella by employing m5C RNA immunoprecipitation followed by a deep-sequencing approach (m5C-RIP-seq). Our data showed that m5C peaks were distributed across the whole mRNA body. Compared with unsporulated oocysts, there were 2813 hypermethylated and 1850 hypomethylated m5C peaks in sporulated oocysts. Generally, a positive correlation between m5C modification and gene expression levels was observed. The mRNA sequencing (RNA-seq) and m5C-RIP-seq data were consistent with the results of the quantitative reverse transcription PCR (RT-qPCR) and methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR), respectively. Gene Ontology (GO) and pathway enrichment analysis predicated diverse biological functions and pathways, including microtubule motor activity, helicase activity, cGMP-PKG signaling pathway, aminoacyl-tRNA biosynthesis, glycolysis/gluconeogenesis, and spliceosome. Meanwhile, stage-specific gene expression signatures of m5C-related regulators were observed. Altogether, our findings reveal the transcriptional significance of m5C modification in E. tenella oocysts, providing resources and clues for further in-depth research.

Single-Cell RNA Sequencing Reveals an Atlas of Hezuo Pig Testis Cells.

Spermatogenesis is a complex biological process crucial for male reproduction and is characterized by intricate interactions between testicular somatic cells and germ cells. Due to the cellular heterogeneity of the testes, investigating different cell types across developmental stages has been challenging. Single-cell RNA sequencing (scRNA-seq) has emerged as a valuable approach for addressing this limitation. Here, we conducted an unbiased transcriptomic study of spermatogenesis in sexually mature 4-month-old Hezuo pigs using 10× Genomics-based scRNA-seq. A total of 16,082 cells were collected from Hezuo pig testes, including germ cells (spermatogonia (SPG), spermatocytes (SPCs), spermatids (SPTs), and sperm (SP)) and somatic cells (Sertoli cells (SCs), Leydig cells (LCs), myoid cells (MCs), endothelial cells (ECs), and natural killer (NK) cells/macrophages). Pseudo-time analysis revealed that LCs and MCs originated from common progenitors in the Hezuo pig. Functional enrichment analysis indicated that the differentially expressed genes (DEGs) in the different types of testicular germ cells were enriched in the PI3K-AKT, Wnt, HIF-1, and adherens junction signaling pathways, while the DEGs in testicular somatic cells were enriched in ECM-receptor interaction and antigen processing and presentation. Moreover, genes related to spermatogenesis, male gamete generation, sperm part, sperm flagellum, and peptide biosynthesis were expressed throughout spermatogenesis. Using immunohistochemistry, we verified several stage-specific marker genes (such as UCHL1, WT1, SOX9, and ACTA2) for SPG, SCs, and MCs. By exploring the changes in the transcription patterns of various cell types during spermatogenesis, our study provided novel insights into spermatogenesis and testicular cells in the Hezuo pig, thereby laying the foundation for the breeding and preservation of this breed.

Design the fusion double-strand RNAs to control two global sap-sucking pests

RNA interference (RNAi) is an effective pest management strategy through silencing the crucial genes in target organisms. However, the effectiveness of targeting a single gene is often limited by the silencing efficiency due to tissue or developmental stage-specific gene expression. Moreover, multiple pests often infest the same crop simultaneously under current ecological conditions. Therefore, a combined strategy of “targeting multiple genes” and “controlling multiple pests” is expected to yield better management results. In this study, homologous genes from two globally sap-sucking pests, the peach aphid (Myzus persicae) and the whitefly (Bemisia tabaci), were screened on a genome-wide scale. Subsequently, RNAi bioassays showed silencing the genes (MpAbd-A, MpH3, MpRpL27a, and MpScr) exhibited high mortalities in both species, which were further selected for designing fusion dsRNAs. These fusion dsRNAs resulted in higher mortalities in both pests than single gene silencing and posed a minimal off-target risk to the predator ladybeetle (Propylaea japonica) based on the sequence analysis. Finally, the tobacco plants expressing the fusion dsRNAs through virus-induced gene silencing (VIGS) technology enhanced the resistance to both pests. In conclusion, this study proposes a novel RNAi-based approach for managing two sap-sucking pests simultaneously.

Comparative transcriptomics analysis reveals stage-specific gene expression profiles associated with gamete formation in Allium sativum L.

Commercial cultivars of garlic, a popular condiment, are sterile, making genetic variation and germplasm innovation of this plant challenging. Understanding mechanism of gamete sterility in garlic and their key regulatory networks is therefore important for fertility restoration. In this work, we conducted a detailed phenotypic analysis of fertile and sterile garlic genotypes and found that enlargement of topset in the inflorescence of sterile genotypes led to abnormal flowers. Additional cytological observations showed that aberrant meiotic cytokinesis in sterile garlic ultimately resulted in pollen degeneration. Transcriptomics analysis of sterile and fertile genotypes identified possible molecular mechanisms underlying gamete abortion. A total of 100 710 differentially expressed genes (DEGs) between the fertile and sterile garlic flowers at three stages of gamete development were identified, many of which were involved in homologous chromosome synapsis during meiosis, MYB transcription factor regulation, ribosome biogenesis and plant hormone signal transduction. Taken together, these results provide insight into the molecular mechanisms and regulatory networks underlying gamete development in garlic and point to a set of candidate genes for further functional characterization.

Epigenomic landscapes during prefrontal cortex development and aging in rhesus.

The prefrontal cortex (PFC) is essential for higher-level cognitive functions. How epigenetic dynamics participates in PFC development and aging is largely unknown. Here, we profiled epigenomic landscapes of rhesus monkey PFCs from prenatal to aging stages. The dynamics of chromatin states, including higher-order chromatin structure, chromatin interaction and histone modifications are coordinated to regulate stage-specific gene transcription, participating in distinct processes of neurodevelopment. Dramatic changes of epigenetic signals occur around the birth stage. Notably, genes involved in neuronal cell differentiation and layer specification are pre-configured by bivalent promoters. We identified a cis-regulatory module and the transcription factors (TFs) associated with basal radial glia development, which was associated with large brain size in primates. These TFs include GLI3, CREB5 and SOX9. Interestingly, the genes associated with the basal radial glia (bRG)-associated cis-element module, such as SRY and SOX9, are enriched in sex differentiation. Schizophrenia-associated single nucleotide polymorphisms are more enriched in super enhancers (SEs) than typical enhancers, suggesting that SEs play an important role in neural network wiring. A cis-regulatory element of DBN1 is identified, which is critical for neuronal cell proliferation and synaptic neuron differentiation. Notably, the loss of distal chromatin interaction and H3K27me3 signal are hallmarks of PFC aging, which are associated with abnormal expression of aging-related genes and transposon activation, respectively. Collectively, our findings shed light on epigenetic mechanisms underlying primate brain development and aging.

Single-cell transcriptomic analysis of immune cell dynamics in the healthy human endometrium

The microenvironment of the endometrial immune system is crucial to the success of placental implantation and healthy pregnancy. However, the functionalities of immune cells across various stages of the reproductive cycle have yet to be fully comprehended. To address this, we conducted advanced bioinformatic analysis on 230,049 high-quality single-cell transcriptomes from healthy endometrial samples obtained during the proliferative, secretory, early pregnancy, and late pregnancy stages. Our investigation has unveiled that proliferative natural killer (NK) cells, a potential source of endometrial NK cells, exhibit the most robust proliferative and differentiation potential during non-pregnant stages. We have also identified similar differentiation trajectories of NK cells originating from proliferative NK cells across four stages. Notably, during early pregnancy, NK cells demonstrate the highest oxidative phosphorylation metabolism activity, and, in conjunction with macrophages and T cells, exhibit the strongest type II interferon response. With spatial transcriptome data, we have discerned that the most robust immune-non-immune interactions are associated with the promotion and inhibition of cell proliferation, differentiation and migration during four stages. Furthermore, we have compiled lists of stage-specific risk genes implicated in reproductive diseases, which hold promise as potential disease biomarkers. Our study provides insights into the dynamics of the endometrial immune microenvironment during different reproductive cycle stages, thus serving as a reference for detecting pathological changes during pregnancy.

Gene Expression and Phenotypic Assessment of Egg Quality across Developmental Stages of Atlantic Cod throughout the Spawning Season.

Egg quality in fishes is commonly determined by fertilisation success and cleavage patterns as a phenotypic outcome of underlying regulatory mechanisms. Although these phenotypic estimators of egg quality are useful in farming conditions, these "good quality" egg batches do not always translate to good larval growth and survival. The identification of genes involved in embryonic development may help find links between genetic factors of maternal origin and egg quality. Herein, the relative expression of seven stage-specific developmental genes of Atlantic cod was analysed using quantitative PCR to understand the function during embryogenesis and its relationship with egg quality. Genes ccnb2 and pvalb1 showed significant differential expression between developmental stages and significant upregulation from blastula and somite stages, respectively. The comparison of spawning batches showed that the relative gene expression of genes ccnb2, acta, tnnt3 and pvalb1 was significantly higher from the middle of the spawning season where phenotypic quality estimators establish the best egg quality. Moreover, a positive significant correlation was observed between quality estimators based on egg morphology and the genetic expression of genes acta and acta1 during somitogenesis. This study suggests that the combination of quality estimators, genetics and batch timing could help optimise reproductive protocols for commercial stocks of Atlantic cod.

A single-cell atlas of chromatin accessibility in mouse organogenesis.

Organogenesis is a highly complex and precisely regulated process. Here we profiled the chromatin accessibility in >350,000 cells derived from 13 mouse embryos at four developmental stages from embryonic day (E) 10.5 to E13.5 by SPATAC-seq in a single experiment. The resulting atlas revealed the status of 830,873 candidate cis-regulatory elements in 43 major cell types. By integrating the chromatin accessibility atlas with the previous transcriptomic dataset, we characterized cis-regulatory sequences and transcription factors associated with cell fate commitment, such as Nr5a2 in the development of gastrointestinal tract, which was preliminarily supported by the in vivo experiment in zebrafish. Finally, we integrated this atlas with the previous single-cell chromatin accessibility dataset from 13 adult mouse tissues to delineate the developmental stage-specific gene regulatory programmes within and across different cell types and identify potential molecular switches throughout lineage development. This comprehensive dataset provides a foundation for exploring transcriptional regulation in organogenesis.

Tolerance to Haemophilus influenzae infection in human epithelial cells: Insights from a primary cell-based model.

Haemophilus influenzae is a human respiratory pathogen and inhabits the human respiratory tract as its only niche. Despite this, the molecular mechanisms that allow H. influenzae to establish persistent infections of human epithelia are not well understood. Here, we have investigated how H. influenzae adapts to the host environment and triggers the host immune response using a human primary cell-based infection model that closely resembles human nasal epithelia (NHNE). Physiological assays combined with dualRNAseq revealed that NHNE from five healthy donors all responded to H. influenzae infection with an initial, 'unproductive' inflammatory response that included a strong hypoxia signature but did not produce pro-inflammatory cytokines. Subsequently, an apparent tolerance to large extracellular and intraepithelial burdens of H. influenzae developed, with NHNE transcriptional profiles resembling the pre-infection state. This occurred in parallel with the development of intraepithelial bacterial populations, and appears to involve interruption of NFκB signalling. This is the first time that large-scale, persistence-promoting immunomodulatory effects of H. influenzae during infection have been observed, and we were able to demonstrate that only infections with live, but not heat-killed H. influenzae led to immunomodulation and reduced expression of NFκB-controlled cytokines such as IL-1β, IL-36γ and TNFα. Interestingly, NHNE were able to re-activate pro-inflammatory responses towards the end of the 14-day infection, resulting in release of IL-8 and TNFα. In addition to providing first molecular insights into mechanisms enabling persistence of H. influenzae in the host, our data further indicate the presence of infection stage-specific gene expression modules, highlighting fundamental similarities between immune responses in NHNE and canonical immune cells, which merit further investigation.

Molecular insights and antibody response to Dr20/22 in dogs naturally infected with Dirofilaria repens

Subcutaneous dirofilariasis, caused by the parasitic nematode Dirofilaria repens, is a growing concern in Europe, affecting both dogs and humans. This study focused on D. repens Dr20/22, a protein encoded by an alt (abundant larval transcript) gene family. While well-documented in L3 larvae of other filariae species, this gene family had not been explored in dirofilariasis. The research involved cloning Dr20/22 cDNA, molecular characterization, and evaluating its potential application in the diagnosis of dirofilariasis. Although Real-Time analysis revealed mRNA expression in both adult worms and microfilariae, the native protein remained undetected in lysates from both developmental stages. This suggests the protein’s specificity for L3 larvae and may be related to a process called SLTS (spliced leader trans-splicing), contributing to stage-specific gene expression. The specificity of the antigen for invasive larvae positions it as a promising early marker for dirofilariasis. However, ELISA tests using sera from infected and uninfected dogs indicated limited diagnostic utility. While further research is required, our findings contribute to a deeper understanding of the molecular and immunological aspects of host-parasite interactions and could offer insights into the parasite's strategies for evading the immune system.

Transcriptomic profiling of gastrointestinal tracts in dairy cattle during lactation reveals molecular adaptations for milk synthesis

During lactation, dairy cattle’s digestive tract requires significant adaptations to meet the increased nutrient demands for milk production. As we attempt to improve milk-related traits through selective pressure, it is crucial to understand the biological functions of the epithelia of the rumen, small intestine, and colonic tissues in response to changes in physiological state driven by changes in nutrient demands for milk synthesis. In this study, we obtained a total of 108 transcriptome profiles from three tissues (epithelia of the colon, duodenum, and rumen) of five Holstein cows, spanning eight time points from the early, mid, late lactation periods to the dry period. On average 97.06% of reads were successfully mapped to the reference genome assembly ARS-UCD1.2. We analyzed 27,607 gene expression patterns at multiple periods, enabling direct comparisons within and among tissues during different lactation stages, including early and peak lactation. We identified 1645, 813, and 2187 stage-specific genes in the colon, duodenum, and rumen, respectively, which were enriched for common or specific biological functions among different tissues. Time series analysis categorized the expressed genes within each tissue into four clusters. Furthermore, when the three tissues were analyzed collectively, 36 clusters of similarly expressed genes were identified. By integrating other comprehensive approaches such as gene co-expression analyses, functional enrichment, and cell type deconvolution, we gained profound insights into cattle lactation, revealing tissue-specific characteristics of the gastrointestinal tract and shedding light on the intricate molecular adaptations involved in nutrient absorption, immune regulation, and cellular processes for milk synthesis during lactation.

The Toxoplasma gondii F-Box Protein L2 Functions as a Repressor of Stage Specific Gene Expression.

Toxoplasma gondii is a foodborne pathogen that can cause severe and life-threatening infections in fetuses and immunocompromised patients. Felids are its only definitive hosts, and a wide range of animals, including humans, serve as intermediate hosts. When the transmissible bradyzoite stage is orally ingested by felids, they transform into merozoites that expand asexually, ultimately generating millions of gametes for the parasite sexual cycle. However, bradyzoites in intermediate hosts differentiate exclusively to disease-causing tachyzoites, which rapidly disseminate throughout the host. Though tachyzoites are well-studied, the molecular mechanisms governing transitioning between developmental stages are poorly understood. Each parasite stage can be distinguished by a characteristic transcriptional signature, with one signature being repressed during the other stages. Switching between stages require substantial changes in the proteome, which is achieved in part by ubiquitination. F-box proteins mediate protein poly-ubiquitination by recruiting substrates to SKP1, Cullin-1, F-Box protein E3 ubiquitin ligase (SCF-E3) complexes. We have identified an F-box protein named Toxoplasma gondii F-Box Protein L2 (TgFBXL2), which localizes to distinct perinucleolar sites. TgFBXL2 is stably engaged in an SCF-E3 complex that is surprisingly also associated with a COP9 signalosome complex that negatively regulates SCF-E3 function. At the cellular level, TgFBXL2-depleted parasites are severely defective in centrosome replication and daughter cell development. Most remarkable, RNAseq data show that TgFBXL2 conditional depletion induces the expression of stage-specific genes including a large cohort of genes necessary for sexual commitment. Together, these data suggest that TgFBXL2 is a latent guardian of stage specific gene expression in Toxoplasma and poised to remove conflicting proteins in response to an unknown trigger of development.

Transcriptional control of the Cryptosporidium life cycle.

The parasite Cryptosporidium is aleading agent of diarrhoeal disease in young children, and a cause and consequence of chronic malnutrition1,2. There are no vaccines and only limited treatment options3. The parasite infects enterocytes, in which it engages in asexual and sexual replication4, both of which are essential to continued infection and transmission. However, their molecular mechanisms remain largely unclear5. Here we use single-cell RNA sequencing to reveal the gene expression programme of the entire Cryptosporidium parvum life cycle in culture and in infected animals. Diverging from the prevailing model6, we find support for only three intracellular stages: asexual type-I meronts, male gamonts and female gametes. We reveal a highly organized program for the assembly of components at each stage. Dissecting the underlying regulatory network, we identify the transcription factor Myb-M as the earliest determinant of male fate, in an organism that lacks genetic sex determination. Conditional expression of this factor overrides the developmental program and induces widespread maleness, while conditional deletion ablates male development. Both have a profound impact on the infection. A large set of stage-specific genes now provides the opportunity to understand, engineer and disrupt parasite sex and life cycle progression to advance the development of vaccines and treatments.

The Roles of N6-Methyladenosine Modification and Its Regulators in Male Reproduction

Infertility affects an estimated 10 to 15 percent of couples worldwide, with approximately half of the cases attributed to male-related issues. Most men diagnosed with infertility exhibit symptoms such as oligospermia, asthenospermia, azoospermia, and compromised sperm quality. Spermatogenesis is a complex and tightly coordinated process of germ cell differentiation, precisely regulated at transcriptional, posttranscriptional, and translational levels to ensure stage-specific gene expression during the development of spermatogenic cells and normal spermiogenesis. N6-methyladenosine (m6A) stands out as the most prevalent modification on eukaryotic mRNA, playing pivotal roles in various biological processes, including mRNA splicing, transportation, and translation. RNA methylation modification is a dynamic and reversible process primarily mediated by "writers", removed by "erasers", and recognized by "readers". In mammals, the aberrant methylation modification of m6A on mRNA is associated with a variety of diseases, including male infertility. However, the precise involvement of disrupted m6A modification in the pathogenesis of human male infertility remains unresolved. Intriguingly, a significant correlation has been found between the expression levels of m6A regulators in the testis and the severity of sperm concentration, motility, and morphology. Aberrant expression patterns of m6A regulatory proteins have been detected in anomalous human semen samples, including those of oligospermia, asthenozoospermia, and azoospermia. Furthermore, the examination of both sperm samples and testicular tissues revealed abnormal mRNA m6A modification, leading to reduced sperm motility and concentration in infertile men. Consequently, it is hypothesized that dysregulation of m6A modification might serve as an integral link in the mechanism of male infertility. This paper presents a comprehensive review of the recent discoveries regarding the spatial and temporal expression dynamics of m6A regulators in testicular tissues and the correlation between deregulated m6A regulators and human male infertility. Previous studies predominantly utilized constitutive or conditional knockout animal models for testicular phenotypic investigations. However, gene suppression in additional tissues could potentially influence the testis in constitutive knockout models. Furthermore, considering the compromised spermatogenesis observed in constitutive animals, distinguishing between the indirect effects of gene depletion on testicular development and its direct impact on the spermatogenic process is challenging, due to their intricate relationship. Such confounding factors might compromise the validity of the findings. To address this challenge, an inducible and conditional gene knockout model may serve as a superior approach. To date, nearly all reported studies have concentrated solely on the level changes of m6A and its regulators in germs cells, while the understanding of the function of m6A modification in testicular somatic cells remains limited. Testicular somatic cells, including peritubular myoid cells, Sertoli cells, and Leydig cells, play indispensable roles during spermatogenesis. Hence, comprehensive exploration of m6A modification within these cells as an additional crucial regulatory mechanism is warranted. In addition, exploration into the presence of unique methylation mechanisms or m6A regulatory factors within the testes is warranted. To elucidate the role of m6A modification in germ cells and testicular somatic cells, detailed experimental strategies need to be implemented. Among them, manipulation of the levels of key enzymes involved in m6A methylation and demethylation might be the most effective approach. Moreover, comprehensive analysis of the gene expression profiles involved in various signaling pathways, such as Wnt/β-catenin, Ras/MAPK, and Hippo, in m6A-modified germ cells and testicular somatic cells can provide more insight into its regulatory role in the spermatogenesis process. Further research in this area could provide valuable insights for developing innovative strategies to treat male infertility. Finally, considering the mitigation impact of m6A imbalance regulation on disease, investigation concerning whether restoring the equilibrium of m6A modification regulation can restore normal spermatogenesis function is essential, potentially elucidating the pivotal clinical significance of m6A modulation in male infertility.

An ApiAp2 Transcription Factor with a Dispensable Role in Plasmodium berghei Life Cycle.

Malaria parasites have a complex life cycle and undergo replication and population expansion within vertebrate hosts and mosquito vectors. These developmental transitions rely on changes in gene expression and chromatin reorganization that result in the activation and silencing of stage-specific genes. The ApiAp2 family of DNA-binding proteins plays an important role in regulating gene expression in malaria parasites. Here, we characterized the ApiAp2 protein in Plasmodium berghei, which we termed Ap2-D. In silico analysis revealed that Ap2-D has three beta-sheets followed by a helix at the C-terminus for DNA binding. Using gene tagging with 3XHA-mCherry, we found that Ap2-D is expressed in Plasmodium blood stages and is present in the parasite cytoplasm and nucleus. Surprisingly, our gene deletion study revealed a completely dispensable role for Ap2-D in the entirety of the P. berghei life cycle. Ap2-D KO parasites were found to grow in the blood successfully and progress through the mosquito midgut and salivary glands. Sporozoites isolated from mosquito salivary glands were infective for hepatocytes and achieved similar patency as WT in mice. We emphasize the importance of genetic validation of antimalarial drug targets before progressing them to drug discovery.