Multiscale microscopy to decipher plant cell structure and dynamics

The New phytologist

2022 Dec 07

Cui, Y;Zhang, X;Li, X;Lin, J;
PMID: 36477856 | DOI: 10.1111/nph.18641

New imaging methodologies with high contrast and molecular specificity allow researchers to analyze dynamic processes in plant cells at multiple scales, from single protein and RNA molecules to organelles and cells, to whole organs and tissues. These techniques produce informative images and quantitative data on molecular dynamics to address questions that cannot be answered by conventional biochemical assays. Here, we review selected microscopy techniques, focusing on their basic principles and applications in plant science, discussing the pros and cons of each technique, and introducing methods for quantitative analysis. This review thus provides guidance for plant scientists in selecting the most appropriate techniques to decipher structures and dynamic processes at different levels, from protein dynamics to morphogenesis.

Gene expression data visualization tool on the o²S²PARC platform

F1000Research

2022 Nov 07

Ben Aribi, H;Ding, M;Kiran, A;
| DOI: 10.12688/f1000research.126840.1

Background: The identification of differentially expressed genes and their associated biological processes, molecular function, and cellular components are important for genetic diseases studies because they present potential biomarkers and therapeutic targets. Methods: In this study, we developed an o²S²PARC template representing an interactive pipeline for the gene expression data visualization and ontologies data analysis and visualization. To demonstrate the usefulness of the tool, we performed a case study on a publicly available dataset. Results: The tool enables users to identify the differentially expressed genes (DEGs) and visualize them in a volcano plot format. The ontologies associated with the DEGs are determined and visualized in barplots. Conclusions: The “Expression data visualization” template is publicly available on the o²S²PARC platform.

Cross-species single-cell transcriptomic analysis reveals factors limiting human Müller glial-derived retinal regeneration

Investigative Ophthalmology & Visual Science

2022 Jan 01

Dhodapkar, R;Martell, D;Calapkulu, E;

RESULTS : After quality control and data integration, 17,401 nuclei were isolated from 26,471 original droplets, derived from macular samples of 4 patients without retinal disease and 3 patients with POAG. The proportion of retinal ganglion cells in glaucomatous retina was significantly lower than that in healthy retina (p=0.024). An activated subpopulation of Müller glia was identified in both healthy and glaucomatous retina by cell clustering. Cross-species analysis comparing zebrafish and humans identified YAP1 activation as a differentiator between zebrafish and human glial activation. Human retinal explants cultured with N3B1P3C demonstrated significant proliferation of GS+ Muller cells (p=0.044).

In Situ Hybridization (ISH) Combined with Immunohistochemistry (IHC) for Co-detection of EGFR RNA and Phosphorylated EGFR Protein in Lung Cancer Tissue

Methods in molecular biology (Clifton, N.J.)

2022 Dec 13

Oliver, A;Hagen, J;Yang, S;Kalyuzhny, AE;
PMID: 36513934 | DOI: 10.1007/978-1-0716-2811-9_14

Detection of phosphorylated proteins in tissue sections using immunohistochemistry (IHC) is a challenging task. The absence of tissue staining may be caused by either a lack of protein expression or a lack of protein activation via its phosphorylation. To address this problem, we employed Integrated Co-detection Workflow (ICW) protocol to analyze lung cancer tissue sections by combining in situ hybridization (ISH) with IHC. The target protein of interest was epidermal growth factor receptor (EGFR, also known as ErbB1 and HER1) which is the founding member of the ErbB family of receptor tyrosine kinases. Using phospho-specific antibodies specific for a phosphorylated site Y1173 of EGFR molecule allowed us to analyze IHC and ISH staining at a single cell level in lung cancer tissue. We have observed both a co-localization of IHC with ISH signals and ISH-positive cells lacking IHC labeling for phosphorylated EGFR. ICW appears to be a very powerful spatial biology technique for accurate localization of cancer cells with phosphorylated/activated and non-phosphorylated/nonactivated proteins.

Di-valent siRNA-mediated silencing of MSH3 blocks somatic repeat expansion in mouse models of Huntington's disease

Molecular therapy : the journal of the American Society of Gene Therapy

2023 May 12

O'Reilly, D;Belgrad, J;Ferguson, C;Summers, A;Sapp, E;McHugh, C;Mathews, E;Boudi, A;Buchwald, J;Ly, S;Moreno, D;Furgal, R;Luu, E;Kennedy, Z;Hariharan, V;Monopoli, K;Yang, XW;Carroll, J;DiFiglia, M;Aronin, N;Khvorova, A;
PMID: 37177784 | DOI: 10.1016/j.ymthe.2023.05.006

Huntington's disease (HD) is a severe neurodegenerative disorder caused by the expansion of the CAG trinucleotide repeat tract in the huntingtin gene. Inheritance of expanded CAG repeats is needed for HD manifestation, but further somatic expansion of the repeat tract in non-dividing cells, particularly striatal neurons, hastens disease onset. Called somatic repeat expansion, this process is mediated by the mismatch repair (MMR) pathway. Among MMR components identified as modifiers of HD onset, MutS homolog 3 (MSH3) has emerged as a potentially safe and effective target for therapeutic intervention. Here, we identify a fully chemically modified short interfering RNA (siRNA) that robustly silences Msh3 in vitro and in vivo. When synthesized in a di-valent scaffold, siRNA-mediated silencing of Msh3 effectively blocked CAG-repeat expansion in the striatum of two HD mouse models without affecting tumor-associated microsatellite instability or mRNA expression of other MMR genes. Our findings establish a promising treatment approach for patients with HD and other repeat expansion diseases.

The anorectic and thermogenic effects of pharmacological lactate in male mice are confounded by treatment osmolarity and co-administered counterions

Nature metabolism

2023 Apr 01

Lund, J;Breum, AW;Gil, C;Falk, S;Sass, F;Isidor, MS;Dmytriyeva, O;Ranea-Robles, P;Mathiesen, CV;Basse, AL;Johansen, OS;Fadahunsi, N;Lund, C;Nicolaisen, TS;Klein, AB;Ma, T;Emanuelli, B;Kleinert, M;Sørensen, CM;Gerhart-Hines, Z;Clemmensen, C;
PMID: 37055619 | DOI: 10.1038/s42255-023-00780-4

Lactate is a circulating metabolite and a signalling molecule with pleiotropic physiological effects. Studies suggest that lactate modulates energy balance by lowering food intake, inducing adipose browning and increasing whole-body thermogenesis. Yet, like many other metabolites, lactate is often commercially produced as a counterion-bound salt and typically administered in vivo through hypertonic aqueous solutions of sodium L-lactate. Most studies have not controlled for injection osmolarity and the co-injected sodium ions. Here, we show that the anorectic and thermogenic effects of exogenous sodium L-lactate in male mice are confounded by the hypertonicity of the injected solutions. Our data reveal that this is in contrast to the antiobesity effect of orally administered disodium succinate, which is uncoupled from these confounders. Further, our studies with other counterions indicate that counterions can have confounding effects beyond lactate pharmacology. Together, these findings underscore the importance of controlling for osmotic load and counterions in metabolite research.

Recent advances in single-cell subcellular sampling

Chemical communications (Cambridge, England)

2023 May 02

Sahota, A;Monteza Cabrejos, A;Kwan, Z;Paulose Nadappuram, B;Ivanov, AP;Edel, JB;
PMID: 37039236 | DOI: 10.1039/d3cc00573a

Recent innovations in single-cell technologies have opened up exciting possibilities for profiling the omics of individual cells. Minimally invasive analysis tools that probe and remove the contents of living cells enable cells to remain in their standard microenvironment with little impact on their viability. This negates the requirement of lysing cells to access their contents, an advancement from previous single-cell manipulation methods. These novel methods have the potential to be used for dynamic studies on single cells, with many already providing high intracellular spatial resolution. In this article, we highlight key technological advances that aim to remove the contents of living cells for downstream analysis. Recent applications of these techniques are reviewed, along with their current limitations. We also propose recommendations for expanding the scope of these technologies to achieve comprehensive single-cell tracking in the future, anticipating the discovery of subcellular mechanisms and novel therapeutic targets and treatments, ultimately transforming the fields of spatial transcriptomics and personalised medicine.

Advances and Challenges in Spatial Transcriptomics for Developmental Biology

Biomolecules

2023 Jan 12

Choe, K;Pak, U;Pang, Y;Hao, W;Yang, X;
PMID: 36671541 | DOI: 10.3390/biom13010156

Development from single cells to multicellular tissues and organs involves more than just the exact replication of cells, which is known as differentiation. The primary focus of research into the mechanism of differentiation has been differences in gene expression profiles between individual cells. However, it has predominantly been conducted at low throughput and bulk levels, challenging the efforts to understand molecular mechanisms of differentiation during the developmental process in animals and humans. During the last decades, rapid methodological advancements in genomics facilitated the ability to study developmental processes at a genome-wide level and finer resolution. Particularly, sequencing transcriptomes at single-cell resolution, enabled by single-cell RNA-sequencing (scRNA-seq), was a breath-taking innovation, allowing scientists to gain a better understanding of differentiation and cell lineage during the developmental process. However, single-cell isolation during scRNA-seq results in the loss of the spatial information of individual cells and consequently limits our understanding of the specific functions of the cells performed by different spatial regions of tissues or organs. This greatly encourages the emergence of the spatial transcriptomic discipline and tools. Here, we summarize the recent application of scRNA-seq and spatial transcriptomic tools for developmental biology. We also discuss the limitations of current spatial transcriptomic tools and approaches, as well as possible solutions and future prospects.

Far from home: the role of glial mRNA localization in synaptic plasticity

RNA (New York, N.Y.)

2022 Nov 28

Gala, DS;Titlow, JS;Teodoro, RO;Davis, I;
PMID: 36442969 | DOI: 10.1261/rna.079422.122

Neurons and glia are highly polarized cells, whose distal cytoplasmic functional subdomains require specific proteins. Neurons have axonal and dendritic cytoplasmic extensions containing synapses requiring mRNA transport and localized translation to regulate synaptic plasticity efficiently. The principles behind these mechanisms are equally attractive for explaining rapid local regulation of distal glial cytoplasmic projections, independent of their cell nucleus. However, in contrast to neurons, this topic has received little experimental attention in glia. Nevertheless, there are many functionally diverse glial sub-types, containing extensive networks of long cytoplasmic projections with likely localized regulation that influence neurons and their synapses. Moreover, glia have many other neuron-like properties, including electrical activity, secretion of gliotransmitters and calcium signaling, influencing for example synaptic transmission, plasticity and axon pruning. Here, we review previous studies concerning glial transcripts with important roles in influencing synaptic plasticity, focusing on a few cases involving localized translation. We discuss a variety of important questions about mRNA transport and localized translation in glia that remain to be addressed using cutting-edge tools already available for neurons.

Sensory specializations drive octopus and squid behaviour

Nature

2023 Apr 01

Kang, G;Allard, CAH;Valencia-Montoya, WA;van Giesen, L;Kim, JJ;Kilian, PB;Bai, X;Bellono, NW;Hibbs, RE;
PMID: 37045917 | DOI: 10.1038/s41586-023-05808-z

The evolution of new traits enables expansion into new ecological and behavioural niches. Nonetheless, demonstrated connections between divergence in protein structure, function and lineage-specific behaviours remain rare. Here we show that both octopus and squid use cephalopod-specific chemotactile receptors (CRs) to sense their respective marine environments, but structural adaptations in these receptors support the sensation of specific molecules suited to distinct physiological roles. We find that squid express ancient CRs that more closely resemble related nicotinic acetylcholine receptors, whereas octopuses exhibit a more recent expansion in CRs consistent with their elaborated 'taste by touch' sensory system. Using a combination of genetic profiling, physiology and behavioural analyses, we identify the founding member of squid CRs that detects soluble bitter molecules that are relevant in ambush predation. We present the cryo-electron microscopy structure of a squid CR and compare this with octopus CRs1 and nicotinic receptors2. These analyses demonstrate an evolutionary transition from an ancestral aromatic 'cage' that coordinates soluble neurotransmitters or tastants to a more recent octopus CR hydrophobic binding pocket that traps insoluble molecules to mediate contact-dependent chemosensation. Thus, our study provides a foundation for understanding how adaptation of protein structure drives the diversification of organismal traits and behaviour.

Spatially resolved transcriptomics: a comprehensive review of their technological advances, applications, and challenges

Journal of genetics and genomics = Yi chuan xue bao

2023 Mar 27

Cheng, M;Jiang, Y;Xu, J;Mentis, AA;Wang, S;Zheng, H;Sahu, SK;Liu, L;Xu, X;
PMID: 36990426 | DOI: 10.1016/j.jgg.2023.03.011

The ability to explore life kingdoms is largely drive by innovations and breakthroughs in technology, from the invention of the microscope 350 years ago to the recent emergence of single cell sequencing, by which the scientific community has been able to visualize life at an unprecedented resolution. Most recently, the Spatially Resolved Transcriptomics (SRT) technologies have filled the gap in probing the spatial or even three-dimensional (3D) organization of the molecular foundation behind the molecular mysteries of life, including the origin of different cellular populations developed from totipotent cells and human diseases. In this review, we introduce recent progress and challenges on SRT from the perspectives of technologies and bioinformatic tools, as well as the representative SRT applications. With the currently fast-moving progress of the SRT technologies and promising results from early adopted research projects, we can foresee the bright future of such new tools in understanding life at the most profound analytical level.

Canonical Wnt signaling regulates soft palate development through mediating ciliary homeostasis

Development (Cambridge, England)

2023 Feb 24

Janečková, E;Feng, J;Guo, T;Han, X;Ghobadi, A;Araujo-Villalba, A;Rahman, MS;Ziaei, H;Ho, TV;Pareek, S;Alvarez, J;Chai, Y;
PMID: 36825984 | DOI: 10.1242/dev.201189

Craniofacial morphogenesis requires complex interactions involving different tissues, signaling pathways, secreted factors, and organelles. The details of these interactions remain elusive. In this study, we have analyzed the molecular mechanisms and homeostatic cellular activities governing soft palate development to improve regenerative strategies for cleft palate patients. We have identified canonical Wnt signaling as a key signaling pathway primarily active in cranial neural crest (CNC)-derived mesenchymal cells surrounding soft palatal myogenic cells. Using Osr2-Cre;β-cateninfl/fl mice, we show that Wnt signaling is indispensable for mesenchymal cell proliferation and subsequently myogenesis through mediating ciliogenesis. Specifically, we have identified that Wnt signaling directly regulates expression of the ciliary gene Ttll3. Impaired ciliary disassembly leads to differentiation defects of mesenchymal cells and indirectly disrupts myogenesis through decreased expression of Dlk1, a mesenchymal cell-derived pro-myogenesis factor. Moreover, we show that siRNA-mediated reduction of Ttll3 expression partly rescues mesenchymal cell proliferation and myogenesis in the palatal explant cultures from Osr2-Cre;β-cateninfl/fl embryos. This study highlights the role of Wnt signaling in palatogenesis through controlling ciliary homeostasis, which establishes a new mechanism for Wnt-regulated craniofacial morphogenesis.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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