Biological systems' quantitative information is extractable through high-content fluorescence microscopy, a technique that integrates the high-throughput method's efficiency. For fixed planarian cells, a modular assay collection is presented, enabling multiplexed biomarker measurements within microwell plates. Protocols for RNA fluorescent in situ hybridization (RNA FISH) and immunocytochemical methods for quantifying proliferating cells, targeting phosphorylated histone H3 and 5-bromo-2'-deoxyuridine (BrdU) incorporated into nuclear DNA, are included. Assay performance remains consistent across planarian sizes, thanks to the tissue's pre-fixation and staining disaggregation into a single-cell suspension. The adoption of high-content microscopy for planarian samples necessitates minimal additional investment, leveraging the existing reagent infrastructure of established whole-mount staining protocols.
Employing whole-mount in situ hybridization (WISH) methods, incorporating colorimetric or fluorescent in situ hybridization (FISH) approaches, allows for the visualization of endogenous RNA. WISH protocols for planarians, particularly those under the model species Schmidtea mediterranea and Dugesia japonica and larger than 5 mm, are well-established and readily available. Nonetheless, the sexual stress experienced by Schmidtea mediterranea, a subject of study for germline development and function, manifests in significantly larger body sizes exceeding 2 centimeters. Owing to insufficient tissue permeabilization, the current whole-mount WISH protocols are not ideal for specimens of this magnitude. For sexually mature Schmidtea mediterranea, measuring 12 to 16 millimeters, a resilient WISH protocol is described, offering a viable approach for transferring the WISH method to other large planarian species.
The visualization of transcripts through in situ hybridization (ISH) has been a crucial technique in investigating molecular pathways, ever since planarian species were adopted as laboratory models. Various aspects of planarian regeneration, as elucidated by ISH studies, span anatomical specifics of different organs, the distribution of stem cell populations, and the associated signaling pathways. selleck Single-cell and high-throughput sequencing approaches have enabled a more detailed examination of gene expression and cellular lineages. A powerful tool for understanding finer distinctions in intercellular transcriptional patterns and intracellular mRNA distribution is single-molecule fluorescent in situ hybridization (smFISH). This technique, in addition to providing an overall understanding of expression patterns, allows for the detailed analysis of individual transcripts, thereby enabling quantification. Hybridization of individual oligonucleotides, each tagged with a single fluorescent label and complementary to the target transcript, constitutes the means of achieving this. A signal is manifested only when labelled oligonucleotides, focused on the same transcript, hybridize, thus mitigating background and off-target issues. Furthermore, the procedure involves significantly fewer steps than the conventional ISH protocol, thereby optimizing time efficiency. The preparation of whole-mount Schmidtea mediterranea specimens, including tissue preparation, probe synthesis, and smFISH procedures, is augmented by immunohistochemistry.
Whole-mount in situ hybridization stands as a powerful tool for visualizing specific mRNA molecules and subsequently unraveling complex biological inquiries. Within planarian research, this technique is highly valuable, for instance, in charting gene expression throughout the entire regeneration process, and for scrutinizing the results of silencing any gene to establish its specific functions. This chapter comprehensively details the WISH protocol, a standard procedure in our lab, employing a digoxigenin-labeled RNA probe and visualized using NBT-BCIP. Building on the work of Currie et al. (EvoDevo 77, 2016), this protocol represents a synthesis of modifications introduced by several laboratories in recent years to the initial protocol from Kiyokazu Agata's lab in 1997. This common NBT-BCIP WISH protocol, or its minor variations, used in the planarian field, needs a nuanced approach based on our findings. The timing and technique of NAC treatment need to be adjusted based on the specific gene under investigation, especially with regards to epidermal markers.
Visualizing a wide range of genetic expression and tissue composition shifts within Schmidtea mediterranea, using multiple molecular tools simultaneously, has consistently been a highly sought-after capability. The standard techniques for detection commonly include fluorescent in situ hybridization (FISH) and immunofluorescence (IF). To achieve simultaneous execution of both protocols, a novel technique is proposed, which can be augmented by fluorescent-conjugated lectin staining to broaden the spectrum of detectable tissues. We provide a novel protocol for lectin fixation to improve signal clarity, necessary for single-cell level resolution studies.
The piRNA pathway in planarian flatworms is directed by three PIWI proteins, identified as SMEDWI-1, SMEDWI-2, and SMEDWI-3, where SMEDWI is an abbreviation for Schmidtea mediterranea PIWI. Planarians' extraordinary regenerative prowess, driven by the interplay of three PIWI proteins and their affiliated small noncoding RNAs (piRNAs), supports tissue homeostasis and, ultimately, ensures the survival of the animal. Next-generation sequencing is an unavoidable requirement for determining the piRNA sequences, which ultimately define the molecular targets recognized by PIWI proteins. Subsequent to the sequencing procedure, the task at hand is to identify and understand the genomic targets and the regulatory potential of the isolated piRNA populations. This bioinformatics analysis pipeline, specifically developed for planarian piRNAs, enables their systematic processing and characterization. The pipeline's processing entails eliminating PCR duplicates marked by unique molecular identifiers (UMIs), and it incorporates an approach for handling piRNA multimapping to varied genomic regions. Our protocol's inclusion of a fully automated pipeline, readily available on GitHub, is noteworthy. By integrating the presented computational pipeline and the piRNA isolation and library preparation protocol detailed in the accompanying chapter, researchers gain the ability to explore the functional role of the piRNA pathway in flatworm biology.
The regenerative prowess and survival of planarian flatworms are intrinsically linked to the presence of piRNAs and SMEDWI (Schmidtea mediterranea PIWI) proteins. The knockdown of SMEDWI proteins severely impacts the specification of planarian germline and stem cell differentiation, causing lethal outcomes. Due to the fact that the molecular targets and biological roles of PIWI proteins are determined by the small RNAs, named piRNAs (PIWI-interacting RNAs), which bind to PIWI proteins, it is vital to study the large quantity of PIWI-bound piRNAs employing next-generation sequencing. To prepare for sequencing, piRNAs bonded to individual SMEDWI proteins must be isolated. otitis media For this purpose, we developed an immunoprecipitation procedure applicable to all planarian SMEDWI proteins. Co-immunoprecipitated piRNAs are made visible using qualitative radioactive 5'-end labeling, a method that accurately detects even trace amounts of small RNA molecules. Subsequently, individual piRNAs undergo a library preparation process meticulously designed for the effective isolation of piRNAs, specifically those with a 2'-O-methyl modification at their 3' ends. stem cell biology The process of next-generation sequencing, using Illumina technology, is applied to the successfully created piRNA libraries. As detailed in the accompanying manuscript, the obtained data underwent analysis.
Transcriptomic information, derived from RNA sequencing, has become a highly effective means of reconstructing the evolutionary connections between species. Although the core steps of phylogenetic inference remain similar when moving from analyses with limited molecular markers to those using transcriptomes (including nucleic acid extraction and sequencing, sequence manipulation, and tree inference), each step exhibits notable differences. High quality and quantity are indispensable attributes of the extracted RNA. Although some organisms are uncomplicated to work with, handling others, especially those with a smaller physique, might present considerable difficulties. Importantly, the substantial rise in the amount of collected sequences necessitates increased computational power for both handling the sequences and deriving the subsequent phylogenies. It is no longer possible to analyze transcriptomic data on personal computers or with local graphical programs. The implication of this is a heightened demand for researchers' bioinformatic skills. When deducing phylogenetic relationships using transcriptomic data, the genomic traits specific to each organism group, like heterozygosity levels and base composition percentages, require attention.
Geometric understanding, a fundamental mathematical skill developed during early childhood, is vital for future mathematical progression; however, there is a surprising absence of direct research on the contributing factors to kindergarteners' nascent geometric knowledge. Research into the cognitive underpinnings of geometric knowledge employed a modified pathways model in mathematics with a sample of 99 Chinese kindergarten children, aged 5-7. Linguistic abilities, visual-spatial processing, and quantitative knowledge were integrated within hierarchical multiple regression models. Visual perception, phonological awareness, and rapid automatized naming, factors within linguistic abilities, demonstrated significant predictive power for geometric knowledge variation, when accounting for the effects of age, sex, and nonverbal intelligence. The attainment of geometric skills was not noticeably preceded by quantitative knowledge assessments employing dot comparison or number comparison. The findings reveal that kindergarten children's geometric knowledge is predominantly a product of their visual perception and language abilities, not their quantitative knowledge.