smFISH

Different signaling mechanisms concur to ensure robust tissue patterning and cell fate instruction during animal development. Most of these mechanisms rely on signaling proteins that are produced, transported, and detected. The spatiotemporal dynamics of signaling molecules are largely unknown, yet they determine signal activity's spatial range and time frame. Here, we use the Caenorhabditis elegans embryo to study how Wnt ligands, an evolutionarily conserved family of signaling proteins, dynamically organize to establish cell polarity in a developing tissue...

Single molecule fluorescence in situ hybridisation (smFISH) has become a valuable tool to investigate the mRNA expression of single cells. However, it requires a considerable amount of programming expertise to use currently available open-source analytical software packages to extract and analyse quantitative data about transcript expression. Here, we present FISHtoFigure, a new software tool developed specifically for the analysis of mRNA abundance and co-expression in QuPath-quantified, multi-labelled smFISH data...

The fovea is a small region within the central retina that is responsible for our high acuity daylight vision. Chickens also have a high acuity area (HAA), and are one of the few species that enables studies of the mechanisms of HAA development, due to accessible embryonic tissue and methods to readily perturb gene expression. To enable such studies, we characterized the development of the chick HAA using single molecule fluorescent in situ hybridization (smFISH), along with more classical methods. We found that Fgf8 provides a molecular marker for the HAA throughout development and into adult stages, allowing studies of the cellular composition of this area over time...

Single-molecule fluorescence in situ hybridization (smFISH) represents a promising approach for the quantitative analysis of nucleic acid biomarkers in clinical tissue samples. However, low signal intensity and high background noise are complications that arise from diagnostic pathology when performed with smFISH-based RNA imaging in formalin-fixed paraffin-embedded (FFPE) tissue specimens. Moreover, the associated complex procedures can produce uncertain results and poor image quality. Herein, by combining the high specificity of split DNA probes with the high signal readout of ZnCdSe/ZnS quantum dot (QD) labeling, we introduce QD split-FISH, a high-brightness smFISH technology, to quantify the expression of mRNA in both cell lines and clinical FFPE tissue samples of breast cancer and lung squamous carcinoma...

Nuclear architecture is a potential regulator of gene expression in eukaryotic cells. Studies connecting nuclear architecture to gene expression are often population-averaged and do not report on the cell-level heterogeneity in genome organization and associated gene expression. In this report we present a simple way to combine fluorescence in situ hybridization (FISH)-based detection of DNA, with single-molecule RNA FISH (smFISH) and immunofluorescence (IF), while also preserving the three-dimensional (3D) nuclear architecture of a cell...

The secondary and tertiary structures of RNA play a vital role in the regulation of biological reactions. These structures have been experimentally studied through in vivo and in vitro analyses, and in silico models have become increasingly accurate in predicting them. Recent technologies have diversified RNA structure predictions, from the earliest thermodynamic and molecular dynamic-based RNA structure predictions to deep learning-based conformation predictions in the past decade. While most research on RNA structure prediction has focused on short non-coding RNAs, there has been limited research on predicting the conformation of longer mRNAs...

Single-molecule fluorescence in situ hybridization (smFISH) is a powerful method for the visualization and quantification of individual RNA molecules within intact cells. With its ability to probe gene expression at the single cell and single-molecule level, the technique offers valuable insights into cellular processes and cell-to-cell heterogeneity. Although widely used in the animal field, its use in plants has been limited. Here, we present an experimental smFISH workflow that allows researchers to overcome hybridization and imaging challenges in plants, including sample preparation, probe hybridization, and signal detection...

The inherent stochastic processes governing gene expression give rise to heterogeneity across individual cells, highlighting the importance of single-cell studies. The emergence of single-molecule fluorescent in situ hybridization (smFISH) enabled gene expression analysis at the single-cell level while including the spatial dimension through the visualization and quantification of mRNAs in intact fixed cells. By combining smFISH with immunofluorescence (IF), a comprehensive approach takes shape facilitating the study of mRNAs and proteins to correlate gene expression profiles to different cellular states...

Candida albicans is the most prevalent human fungal pathogen. Its pathogenicity is linked to the ability of C. albicans to reversibly change morphology and to grow as yeast, pseudohyphae, or hyphal cells in response to environmental stimuli. Understanding the molecular regulation controlling those morphological switches remains a challenge that, if solved, could help eradicate C. albicans infections.While numerous studies investigated gene expression changes occurring during C. albicans morphological switches using bulk approaches (e...

In this chapter, we describe in detail how to perform a successful smFISH experiment and how to quantify mRNA transcripts in bacterial cells. The flexibility of the method allows for straightforward adaptation to different bacterial species and experimental conditions. Thanks to the feasibility of the approach, the method can easily be adapted by other laboratories. Finally, we believe that this method has a great potential to generate insights into the complicated life of bacteria.

The pathologic consequences of Coronavirus Disease-2019 (COVID-19) include elevated inflammation and dysregulated vascular functions associated with thrombosis. In general, disruption of vascular homeostasis and ensuing prothrombotic events are driven by activated platelets, monocytes, and macrophages, which form aggregates (thrombi) attached to the endothelium lining of vessel walls. However, molecular pathways underpinning the pathological interactions between myeloid cells and endothelium during COVID-19 remain undefined...

IMPORTANCE: Habenula (Hb) pathophysiology is involved in many neuropsychiatric disorders, including schizophrenia. Deep brain stimulation and pharmacological targeting of the Hb are emerging as promising therapeutic treatments. However, little is known about the cell type-specific transcriptomic organization of the human Hb or how it is altered in schizophrenia. OBJECTIVE: To define the molecular neuroanatomy of the human habenula and identify transcriptomic changes in individuals with schizophrenia compared to neurotypical controls...

Algal blooms drive global biogeochemical cycles of key nutrients and serve as hotspots for biological interactions in the ocean. The massive blooms of the cosmopolitan coccolithophore Emiliania huxleyi are often infected by the lytic E. huxleyi virus (EhV), which is a major mortality agent triggering bloom demise. This multi-annual "boom and bust" pattern of E. huxleyi blooms suggests that coexistence is essential for these host-virus dynamics. To investigate host-virus coexistence, we developed a new model system from an E...

Traditional single-molecule fluorescence in situ hybridization (smFISH) methods for RNA detection often face sensitivity challenges due to the low fluorescence intensity of the probe. Also, short-lived autofluorescence complicates obtaining clear signals from tissue sections. In response, we have developed an smFISH probe using highly grafted lanthanide complexes to address both concentration quenching and autofluorescence background. Our approach involves an oligo PCR incorporating azide-dUTP, enabling conjugation with lanthanide complexes...

Translation of seed stored mRNAs is essential to trigger germination. However, when RNAPII re-engages RNA synthesis during the seed-to-seedling transition has remained in question. Combining csRNA-seq, ATAC-seq and smFISH in Arabidopsis thaliana we demonstrate that active transcription initiation is detectable during the entire germination process. Features of non-coding regulation such as dynamic changes in chromatin accessible regions, antisense transcription, as well as bidirectional non-coding promoters are widespread throughout the Arabidopsis genome...

In the adult mammalian brain, astrocytes are proposed to be the major Sonic Hedgehog (Shh)-responsive cells. However, the sources of the Shh molecule mediating activation of the pathway are still poorly characterized. The present work investigates the distribution and phenotype of cells expressing Shh mRNA in the adult mouse brain. Using single-molecule fluorescent in situ hybridization (smfISH), we report much broader expression of Shh transcripts in almost all brain regions than originally reported. We identify Shh mRNA in HuC/D+ neuronal populations, including GABAergic (glutamic acid decarboxylase 67, Gad67), cholinergic (choline acetyltransferase, ChAT), dopaminergic (tyrosine hydroxylase, TH), nitrergic (neuronal nitric oxide synthase, nNOS), and in a small population of oligodendroglial cells expressing Sox10 and Olig2 mRNA transcription factors...

Encoding sounds with a high degree of temporal precision is an essential task for the inferior colliculus (IC) to perform and maintain the accurate processing of sounds and speech. However, the age-related reduction of GABAergic neurotransmission in the IC interrupts temporal precision and likely contributes to presbycusis. As presbycusis often manifests at high or low frequencies specifically, we sought to determine if the expression of mRNA for glutamic decarboxylase 1 (GAD1) is downregulated non-uniformly across the tonotopic axis or cell size range in the aging IC...

The basement membrane (BM) demarcating epithelial tissues undergoes rapid expansion to accommodate tissue growth and morphogenesis during embryonic development. To facilitate the secretion of bulky BM proteins, their mRNAs are polarized basally in the follicle epithelial cells of the Drosophila egg chamber to position their sites of production close to their deposition. In contrast, we observed the apical rather than basal polarization of all major BM mRNAs in the outer epithelial cells adjacent to the BM of mouse embryonic salivary glands using single-molecule RNA fluorescence in situ hybridization (smFISH)...

Studies of human lung development have focused on epithelial and mesenchymal cell types and function, but much less is known about the developing lung immune cells, even though the airways are a major site of mucosal immunity after birth. An unanswered question is whether tissue-resident immune cells play a role in shaping the tissue as it develops in utero. Here, we profiled human embryonic and fetal lung immune cells using scRNA-seq, smFISH, and immunohistochemistry. At the embryonic stage, we observed an early wave of innate immune cells, including innate lymphoid cells, natural killer cells, myeloid cells, and lineage progenitors...

BACKGROUND & AIMS: The intestinal epithelium interfaces with a diverse milieu of luminal contents while maintaining robust digestive and barrier functions. Facultative intestinal stem cells are cells that survive tissue injury and divide to re-establish the epithelium. Prior studies demonstrate autophagic state as functional marker of facultative intestinal stem cells, but regulatory mechanisms are not known. The current study evaluated a post-transcriptional regulation of autophagy as an important factor for facultative stem cell state and tissue regeneration...