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Current Protocols Oct 2023The rapid succession of events during development poses an inherent challenge to achieve precise synchronization required for rigorous, quantitative phenotypic and...
The rapid succession of events during development poses an inherent challenge to achieve precise synchronization required for rigorous, quantitative phenotypic and genotypic analyses in multicellular model organisms. Drosophila melanogaster is an indispensable model for studying the development and function of higher order organisms due to extensive genome homology, tractability, and its relatively short lifespan. Presently, nine Nobel prizes serve as a testament to the utility of this elegant model system. Ongoing advancements in genetic and molecular tools allow for the underlying mechanisms of human disease to be investigated in Drosophila. However, the absence of a method to precisely age-match tissues during larval development prevents further capitalization of this powerful model organism. Drosophila spends nearly half of its life cycle progressing through three morphologically distinct larval instar stages, during which the imaginal discs, precursors of mature adult external structures (e.g., eyes, legs, wings), grow and develop distinct cell fates. Other tissues, such as the central nervous system, undergo massive morphological changes during larval development. While these three larval stages and subsequent pupal stages have historically been identified based on the number of hours post egg-laying under standard laboratory conditions, a reproducible, efficient, and inexpensive method is required to accurately age-match larvae within the third instar. The third instar stage is of particular interest, as this developmental stage spans a 48-hr window during which larval tissues switch from proliferative to differentiation programs. Moreover, some genetic manipulations can lead to developmental delays, further compounding the need for precise age-matching between control and experimental samples. This article provides a protocol optimized for synchronous staging of Drosophila third instar larvae by colorimetric characterization and is useful for age-matching a variety of tissues for numerous downstream applications. We also provide a brief discussion of the technical challenges associated with successful application of this protocol. © 2023 Wiley Periodicals LLC. Basic Protocol: Synchronization of third instar Drosophila larvae.
Topics: Animals; Humans; Drosophila; Drosophila melanogaster; Larva; Colorimetry; Pupa
PubMed: 37861353
DOI: 10.1002/cpz1.924 -
Current Opinion in Plant Biology Dec 2023Comparative transcriptomics has emerged as a powerful approach that allows us to unravel the genetic basis of organ morphogenesis and its diversification processes... (Review)
Review
Comparative transcriptomics has emerged as a powerful approach that allows us to unravel the genetic basis of organ morphogenesis and its diversification processes during evolution. However, the application of comparative transcriptomics in studying plant morphological diversity addresses challenges such as identifying homologous gene pairs, selecting appropriate developmental stages for comparison, and extracting biologically meaningful networks. Methods such as phylostratigraphy, clustering, and gene co-expression networks are explored to identify functionally equivalent genes, align developmental stages, and uncover gene regulatory relationships. In the current review, we highlight the importance of these approaches in overcoming the complexity of plant genomes, the impact of heterochrony on stage alignment, and the integration of gene networks with additional data for a comprehensive understanding of morphological evolution.
Topics: Biological Evolution; Gene Expression Profiling; Morphogenesis; Gene Regulatory Networks; Plants
PubMed: 37804608
DOI: 10.1016/j.pbi.2023.102474 -
PeerJ 2023Autotoxicity is an intraspecific manifestation of allelopathy in plant species. The specialized metabolites and their derivatives that cause intraspecific allelopathic... (Review)
Review
BACKGROUND
Autotoxicity is an intraspecific manifestation of allelopathy in plant species. The specialized metabolites and their derivatives that cause intraspecific allelopathic inhibition in the plant are known as autotoxic substances. Consequently, autotoxic substances production seriously affects the renewal and stability of ecological communities.
METHODS
This article systematically summarizes the types of autotoxic substances present in different plants. They mainly include phenolic compounds, terpenoids, and nitrogenous organic compounds. Phenolic coumarins are the main autotoxic substances in many plants. Therefore, we also discuss differences in coumarin types and content among plant varieties, developmental stages, and tissue parts, as well as their mechanisms of autotoxicity. In addition, we review the metabolic pathways involved in coumarin biosynthesis, the key enzymes, genes, and transcription factors, as well as factors affecting coumarin biosynthesis.
RESULTS
Coumarin biosynthesis involves three stages: (1) the formation of the coumarin nucleus; (2) acylation, hydroxylation, and cyclization; (3) structural modification. The key enzymes involved in the coumarin nuclear formation stage include PAL, C4H, 4CL, HCT, CAOMT, COSY, F6'H, and CCoAOMT1, and the key genes involved include BGA, CYP450 and MDR, among others. Ortho-hydroxylation is a key step in coumarin biosynthesis and PS, COSY and S8H are the key enzymes involved in this process. Finally, UGTs are responsible for the glycosylation modification of coumarins, and the MaUGT gene may therefore be involved in coumarin biosynthesis.
CONCLUSION
It is important to elucidate the autotoxicity and anabolic mechanisms of coumarins to create new germplasms that produce fewer autotoxic substances.
Topics: Coumarins; Plants; Cytochrome P-450 Enzyme System; Hydroxylation; Secondary Metabolism
PubMed: 38077428
DOI: 10.7717/peerj.16508 -
Frontiers in Cell and Developmental... 2023Morphological phenotyping of the mouse embryo is described at neurulation stages, primarily as a guide to evaluating the outcome of whole embryo cultures between... (Review)
Review
Morphological phenotyping of the mouse embryo is described at neurulation stages, primarily as a guide to evaluating the outcome of whole embryo cultures between embryonic days 8.5 and 9.5. During this period, neural tube closure is initiated and progresses to completion in the cranial region. Spinal closure is still underway at the end of the culture period. The focus of this article is particularly on phenotyping that can be performed at the bench, using a stereomicroscope. This involves assessment of embryonic health, through observation and scoring of yolk sac blood circulation, measurement of developmental stage by somite counting, and determination of crown-rump length as a measure of growth. Axial rotation ("turning") can also be assessed using a simple scoring system. Neural tube closure assessment includes: 1) determining whether closure has been initiated at the Closure 1 site; 2) evaluating the complex steps of cranial neurulation including initiation at Closure sites 2 and 3, and completion of closure at the anterior and hindbrain neuropores; 3) assessment of spinal closure by measurement of posterior neuropore length. Interpretation of defects in neural tube closure requires an appreciation of, first, the stages that particular events are expected to be completed and, second, the correspondence between embryonic landmarks, for example, somite position, and the resulting adult axial levels. Detailed embryonic phenotyping, as described in this article, when combined with the versatile method of whole embryo culture, can form the basis for a wide range of experimental studies in early mouse neural development.
PubMed: 37601098
DOI: 10.3389/fcell.2023.1223849 -
Nature Methods Dec 2023During animal development, embryos undergo complex morphological changes over time. Differences in developmental tempo between species are emerging as principal drivers...
During animal development, embryos undergo complex morphological changes over time. Differences in developmental tempo between species are emerging as principal drivers of evolutionary novelty, but accurate description of these processes is very challenging. To address this challenge, we present here an automated and unbiased deep learning approach to analyze the similarity between embryos of different timepoints. Calculation of similarities across stages resulted in complex phenotypic fingerprints, which carry characteristic information about developmental time and tempo. Using this approach, we were able to accurately stage embryos, quantitatively determine temperature-dependent developmental tempo, detect naturally occurring and induced changes in the developmental progression of individual embryos, and derive staging atlases for several species de novo in an unsupervised manner. Our approach allows us to quantify developmental time and tempo objectively and provides a standardized way to analyze early embryogenesis.
Topics: Animals; Deep Learning; Embryonic Development; Biological Evolution; Temperature
PubMed: 37996754
DOI: 10.1038/s41592-023-02083-8 -
Fly Dec 2023Valosin-containing protein (VCP) is a versatile and ubiquitously expressed AAA+ ATPase that regulates multiple stages of spermatogenesis. While VCP has documented roles...
Valosin-containing protein (VCP) is a versatile and ubiquitously expressed AAA+ ATPase that regulates multiple stages of spermatogenesis. While VCP has documented roles in mitotic spermatogonia and meiotic spermatocytes, it is also highly expressed in post-meiotic spermatids, suggesting potential late-stage developmental functions as well. However, tools to assess late-stage activities of pleiotropic spermatogenesis genes such as are lacking. Available germline-specific Gal4 drivers activate in stem cells or spermatogonia; consequently, knocking down using one of these drivers disrupts or blocks early germ-cell development, precluding analysis of VCP in later stages. A Gal4 driver that activates later in development, such as at the meiotic spermatocyte stage, may permit functional analyses of VCP and other factors in post-meiotic stages. Here, we describe a germline-specific Gal4 driver, Rbp4-Gal4, which drives transgene expression beginning in the early spermatocyte stage. We find that Rbp4-Gal4-driven knockdown of causes defects in spermatid chromatin condensation and individualization without affecting earlier developmental stages. Interestingly, the defect in chromatin condensation appears linked to errors in the histone-to-protamine transition, a key event in spermatid development. Overall, our study reveals roles for VCP in spermatid development and establishes a powerful tool to dissect the functions of pleiotropic spermatogenesis genes.
Topics: Male; Animals; Spermatids; Valosin Containing Protein; Spermatogenesis; Meiosis; Drosophila; Chromatin
PubMed: 37436409
DOI: 10.1080/19336934.2023.2234795 -
Nature Jan 2024Sexual reproduction of Toxoplasma gondii, confined to the felid gut, remains largely uncharted owing to ethical concerns regarding the use of cats as model organisms....
Sexual reproduction of Toxoplasma gondii, confined to the felid gut, remains largely uncharted owing to ethical concerns regarding the use of cats as model organisms. Chromatin modifiers dictate the developmental fate of the parasite during its multistage life cycle, but their targeting to stage-specific cistromes is poorly described. Here we found that the transcription factors AP2XII-1 and AP2XI-2 operate during the tachyzoite stage, a hallmark of acute toxoplasmosis, to silence genes necessary for merozoites, a developmental stage critical for subsequent sexual commitment and transmission to the next host, including humans. Their conditional and simultaneous depletion leads to a marked change in the transcriptional program, promoting a full transition from tachyzoites to merozoites. These in vitro-cultured pre-gametes have unique protein markers and undergo typical asexual endopolygenic division cycles. In tachyzoites, AP2XII-1 and AP2XI-2 bind DNA as heterodimers at merozoite promoters and recruit MORC and HDAC3 (ref. ), thereby limiting chromatin accessibility and transcription. Consequently, the commitment to merogony stems from a profound epigenetic rewiring orchestrated by AP2XII-1 and AP2XI-2. Successful production of merozoites in vitro paves the way for future studies on Toxoplasma sexual development without the need for cat infections and holds promise for the development of therapies to prevent parasite transmission.
Topics: Animals; Cats; Humans; Chromatin; Disease Models, Animal; Epigenesis, Genetic; In Vitro Techniques; Life Cycle Stages; Merozoites; Nuclear Proteins; Promoter Regions, Genetic; Protozoan Proteins; Toxoplasma; Toxoplasmosis; Transcription, Genetic
PubMed: 38093015
DOI: 10.1038/s41586-023-06821-y -
Horticulture Research Oct 2023Poplar is an important afforestation and urban greening species. Poplar leaf development occurs in stages, from young to mature and then from mature to senescent; these...
Poplar is an important afforestation and urban greening species. Poplar leaf development occurs in stages, from young to mature and then from mature to senescent; these are accompanied by various phenotypic and physiological changes. However, the associated transcriptional regulatory network is relatively unexplored. We first used principal component analysis to classify poplar leaves at different leaf positions into two stages: developmental maturity (the stage of maximum photosynthetic capacity); and the stage when photosynthetic capacity started to decline and gradually changed to senescence. The two stages were then further subdivided into five intervals by gene expression clustering analysis: young leaves, the period of cell genesis and functional differentiation (L1); young leaves, the period of development and initial formation of photosynthetic capacity (L3-L7); the period of maximum photosynthetic capacity of functional leaves (L9-L13); the period of decreasing photosynthetic capacity of functional leaves (L15-L27); and the period of senescent leaves (L29). Using a weighted co-expression gene network analysis of regulatory genes, high-resolution spatiotemporal transcriptional regulatory networks were constructed to reveal the core regulators that regulate leaf development. Spatiotemporal transcriptome data of poplar leaves revealed dynamic changes in genes and miRNAs during leaf development and identified several core regulators of leaf development, such as GRF5 and MYB5. This in-depth analysis of transcriptional regulation during leaf development provides a theoretical basis for exploring the biological basis of the transcriptional regulation of leaf development and the molecular design of breeding for delaying leaf senescence.
PubMed: 37899951
DOI: 10.1093/hr/uhad186 -
Nature Protocols Nov 2023RNA-sequencing (RNA-seq) provides invaluable knowledge on developmental pathways and the effects of mutant phenotypes. Plant reproductive cells have traditionally been... (Review)
Review
RNA-sequencing (RNA-seq) provides invaluable knowledge on developmental pathways and the effects of mutant phenotypes. Plant reproductive cells have traditionally been difficult to isolate for genomics because they are rare and often deeply embedded within somatic tissues. Here, we present a protocol to isolate single maize meiocytes and pollen grains for RNA-seq. We discuss how to identify and isolate each sample type under a microscope, prepare RNA-seq libraries and analyze the data. This technique has several advantages over alternative methods, combining the ability to target specific rare cell types while resolving cell-to-cell heterogeneity with single-cell RNA-seq. The technique is compatible with minute amounts of starting material (e.g., a single anther), making it possible to collect dense time courses. Furthermore, developmentally synchronized anthers are saved for microscopy, allowing staging to be performed in parallel with expression analysis. Up to 200 cells can be collected in 4-5 h by someone proficient in tissue dissection, and library preparation can be completed in 2 d by researchers experienced in molecular biology and genomics. This protocol will facilitate research on plant reproduction, providing insights critical to plant breeding, genetics and agriculture.
Topics: Zea mays; Single-Cell Gene Expression Analysis; Genomics; Pollen; Phenotype
PubMed: 37783945
DOI: 10.1038/s41596-023-00889-6 -
Cells & Development Sep 2023Ceramide induces autophagy upon starvation via downregulation of nutrient transporters. To elucidate the mechanism by which starvation regulates autophagy in mouse...
Ceramide induces autophagy upon starvation via downregulation of nutrient transporters. To elucidate the mechanism by which starvation regulates autophagy in mouse embryos, the present study investigated nutrient transporter expression and the effect of C2-ceramide on in vitro embryo development, apoptosis, and autophagy. The transcript levels of the glucose transporters Glut1 and Glut3 were high at the 1- and 2-cell stages, and gradually decreased at the morula and blastocyst (BL) stages. Similarly, expression of the amino acid transporters L-type amino transporter-1 (LAT-1) and 4F2 heavy chain (4F2hc) gradually decreased from the zygote to the BL stage. Upon ceramide treatment, expression of Glut1, Glut3, LAT-1, and 4F2hc was significantly reduced at the BL stage, while expression of the autophagy-related genes Atg5, LC3, and Gabarap and synthesis of LC3 were significantly induced. Ceramide-treated embryos exhibited significantly reduced developmental rates and total cell numbers per blastocyst, and increased levels of apoptosis and expression of Bcl2l1 and Casp3 at the BL stage. Ceramide treatment significantly decreased the average mitochondrial DNA copy number and mitochondrial area at the BL stage. In addition, ceramide treatment significantly decreased mTOR expression. These results suggest that ceramide-induced autophagy promotes apoptosis by following downregulation of nutrient transporters during mouse embryogenesis.
Topics: Pregnancy; Female; Mice; Animals; Ceramides; Glucose Transporter Type 1; Glucose Transporter Type 3; Embryonic Development; Membrane Transport Proteins; Autophagy
PubMed: 37271244
DOI: 10.1016/j.cdev.2023.203859