Generation and characterization of PDGFRα-GFP knock-in mice for visualization of PDGFRα+ fibroblasts in vivo.

The platelet-derived growth factor (PDGF) and its receptor, PDGFRα, are critical for tissue development and injury repair. To track PDGFRα-expressing cells in vivo, we generated a knock-in mouse line that expresses green fluorescent protein (GFP) under the control of the PDGFRα promoter. This genetic tool enabled us to detect PDGFRα expression in various organs during both neonatal and adult stages. Additionally, we confirmed the correlation between endogenous PDGFRα and transgenic PDGFRα expression using mouse injury models, showing the potential of this genetic reporter for studying PDGFRα-mediated signaling pathways and developing therapeutic strategies. Overall, the PDGFRα-GFP knock-in mouse line serves as a valuable tool for investigating the biology of PDGFRα and its role in normal development and disease.

Zebrafish (Danio rerio) larvae as a model for real-time studies of propagating VHS virus infection, tissue tropism and neutrophil activity.

Viral haemorrhagic septicaemia virus (VHSV) is a negative-sense single-stranded RNA virus that infects more than 140 different fish species. In this study, zebrafish larvae were employed as in vivo model organisms to investigate progression of disease, the correlation between propagation of the infection and irreversibility of disease, cell tropism and in situ neutrophil activity towards the VHSV-infected cells. A recombinant VHSV strain, encoding "tomato" fluorescence (rVHSV-Tomato), was used in zebrafish to be able to follow the progress of the infection in the live host in real-time. Two-day-old zebrafish larvae were injected into the yolk sac with the recombinant virus. The virus titre peaked 96 hr post-infection in zebrafish larvae kept at 18°C, and correlated with 33% mortality and high morbidity among the larvae. By utilizing the transgenic zebrafish line Tg(fli1:GFP)y1 with fluorescently tagged endothelial cells, we were able to demonstrate that the virus initially infected endothelial cells lining the blood vessels. By observing the rVHSV-Tomato infection in the neutrophil reporter zebrafish line Tg(MPX:eGFP)i114 , we inferred that only a subpopulation of the neutrophils responded to the virus infection. We conclude that the zebrafish larvae are suitable for real-time studies of VHS virus infections, allowing in vivo dissection of host-virus interactions at the whole organism level.

Abscisic acid and other plant hormones: Methods to visualize distribution and signaling.

The exploration of plant behavior on a cellular scale in a minimal invasive manner is key to understanding plant adaptations to their environment. Plant hormones regulate multiple aspects of growth and development and mediate environmental responses to ensure a successful life cycle. To monitor the dynamics of plant hormone actions in intact tissue, we need qualitative and quantitative tools with high temporal and spatial resolution. Here, we describe a set of biological instruments (reporters) for the analysis of the distribution and signaling of various plant hormones. Furthermore, we provide examples of their utility for gaining novel insights into plant hormone action with a deeper focus on the drought hormone abscisic acid.

Multicolor 19F magnetic resonance imaging: A promising medical technique for in vivo visualization of multiple biological targets.

Driven by the needs of precision medicine, current imaging techniques are under continuous development to offer more accurate and comprehensive information beyond traditional macroscopic anatomical images. Multispectral color-coded (multicolor) 19F magnetic resonance imaging (MRI) is receiving increasing attention owing to its capability for visualizing quantitative and multiplexed molecular information during various biological processes. The chemical design and preparation of 19F probes lie at the core of multicolor 19F MRI since their performance dominates the accomplishment of this technique. Herein, the working principles of multicolor 19F MRI are briefly introduced. Recent progress on multicolor 19F MRI probes for simultaneous in vivo visualization of multiple biological targets is summarized. Finally, current challenges and potential solutions in this fast-developing field are discussed.

Activatable 19 F MRI Nanoprobes for Visualization of Biological Targets in Living Subjects.

Visualization of biological targets such as crucial cells and biomolecules in living subjects is critical for the studies of important biological processes. Though 1 H magnetic resonance imaging (MRI) has demonstrated its power in offering detailed anatomical and pathological information, its capacity for in vivo tracking of biological targets is limited by the high biological background of 1 H. 19 F distinguishes itself from its competitors as an exceptional complement to 1 H in MRI through its high sensitivity, low biological background, and broad chemical shift range. The specificity and sensitivity of 19 F MRI can be further boosted with activatable nanoprobes. The advantages of 19 F MRI with activatable nanoprobes enable in vivo detection and imaging at the cellular or even molecular level in deep tissues, rendering this technique appealing as a potential solution for visualization of biological targets in living subjects. Here, recent progress over the past decades on activatable 19 F MRI nanoprobes made from three major 19 F-containing compounds, as well as present challenges and potential opportunities, are summarized to provide a panoramic prospective for the people who are interested in this emerging and exciting field.

Trimolecular Fluorescence Complementation (TriFC) Assay for Visualization of RNA-Protein Interaction in Plants.

RNA-protein interactions play important roles in various eukaryotic biological processes. Molecular imaging of subcellular localization of RNA-protein complexes in plants is critical for understanding these interactions. However, methods to image RNA-protein interactions in living plants have not yet been developed until now. Recently, we have developed a trimolecular fluorescence complementation (TriFC) system for in vivo visualization of RNA-protein interaction by transient expression in tobacco leaves. In this method, we combined conventional bimolecular fluorescence complementation (BiFC) system with the MS2 system (phage MS2 coat protein [MCP] and its binding RNA sequence [MS2 sequence]) to tag lncRNA. Target RNA is tagged with 6xMS2, and MCP and RNA-binding protein are fused with YFP fragments. DNA constructs encoding such fusion RNA and proteins are infiltrated into tobacco leaves with Agrobacterium suspensions. RNA-protein interaction in vivo is observed by confocal microscopy.

A Simple Method for Visualization of Locus-Specific H4K20me1 Modifications in Living Caenorhabditis elegans Single Cells.

Recently, advances in next-generation sequencing technologies have enabled genome-wide analyses of epigenetic modifications; however, it remains difficult to analyze the states of histone modifications at a single-cell resolution in living multicellular organisms because of the heterogeneity within cellular populations. Here we describe a simple method to visualize histone modifications on the specific sequence of target locus at a single-cell resolution in living Caenorhabditis elegans, by combining the LacO/LacI system and a genetically-encoded H4K20me1-specific probe, "mintbody". We demonstrate that Venus-labeled mintbody and mTurquoise2-labeled LacI can co-localize on an artificial chromosome carrying both the target locus and LacO sequences, where H4K20me1 marks the target locus. We demonstrate that our visualization method can precisely detect H4K20me1 depositions on the her-1 gene sequences on the artificial chromosome, to which the dosage compensation complex binds to regulate sex determination. The degree of H4K20me1 deposition on the her-1 sequences on the artificial chromosome correlated strongly with sex, suggesting that, using the artificial chromosome, this method can reflect context-dependent changes of H4K20me1 on endogenous genomes. Furthermore, we demonstrate live imaging of H4K20me1 depositions on the artificial chromosome. Combined with ChIP assays, this mintbody-LacO/LacI visualization method will enable analysis of developmental and context-dependent alterations of locus-specific histone modifications in specific cells and elucidation of the underlying molecular mechanisms.