Probes for INS

Fluorescence in situ hybridization (FISH) has become an important tool in laboratory experimentation by providing a qualitative or semi-quantitative technique to detect nucleic acids across different sample types and species. It also serves as a promising platform for the discovery of novel RNA biomarkers and the development of molecular diagnostic assays. While technologies to detect hundreds or thousands of gene transcripts in situ with single-cell resolution are rapidly coming online, smaller scale FISH analysis continues to be highly useful in neuroscience research. In this chapter, we describe a robust, relatively fast and low cost, turnkey in situ hybridization technology (ISH) to identify one or more RNA targets together with immunohistochemical analyses. Specifically, we present a customized version of the protocol that works particularly well for spinal cord and primary sensory ganglia tissues.

The milestone achievement of reprogramming a human somatic cell into a pluripotent stem cell by Yamanaka and Takahashi in 2007 has changed the stem cell research landscape tremendously. Their discovery opened the unprecedented opportunity to work with human-induced pluripotent stem cells and the differentiated progeny thereof, without major ethical restrictions. Additionally, the new method offers the possibility to generate pluripotent stem cells from patients with various genetic diseases which is of great importance (a) to understand the basic mechanisms of a specific disease in a human cellular context and (b) to help find suitable therapies for the persons concerned. In individual cases, this can even help to develop personalized treatment options. Chronic pain is a disease that affects roughly one in five people worldwide, but its onset is rarely based upon genetic alterations. Nevertheless, the work with sensory-like neurons derived from human pluripotent stem cells has become a more widely used tool also in the field of pain research, as during the past years several differentiation procedures have been published that describe the generation of different types of sensory-like neurons and their useful contribution to studying mechanisms of sensitization. Especially also to complement and verify cellular and molecular mechanisms identified in rodent model systems, the model of choice for decades. Although a sole cellular system is not able to mimic a disease as complex as pain, it is a valid tool to understand basic mechanisms of sensitization in specific subsets of human neurons that might be at the onset of the disease. In addition, the creativity of basic researchers and the more and more advanced available technologies will most likely find ways to implement the derived human cells in more complex networks. In this chapter, I want to introduce a selection of published differentiation strategies that result in the generation of human sensory-like neurons. Additionally, I will point out some studies whose results helped to further understand pain-related mechanisms and which were conducted using the aforementioned differentiation procedures.

Heterotopic ossification (HO) is one of the most intractable conditions following injury to the musculoskeletal system. In recent years, much attention has been paid to the role of lncRNA in musculoskeletal disorders, but its role in HO was still unclear. Therefore, this study attempted to determine the role of lncRNA MEG3 in the formation of post-traumatic HO and further explore the underlying mechanisms.On the basis of high-throughput sequencing and qPCR validation, elevated expression of the lncRNA MEG3 was shown during traumatic HO formation. Accordingly, in vitro experiments demonstrated that lncRNA MEG3 promoted aberrant osteogenic differentiation of tendon-derived stem cells (TDSCs). Mechanical exploration through RNA pulldown, luciferase reporter gene assay and RNA immunoprecipitation assay identified the direct binding relationship between miR-129-5p and MEG3, or miR-129-5p and TCF4. Further rescue experiments confirmed the miR-129-5p/TCF4/β-catenin axis to be downstream molecular cascade responsible for the osteogenic-motivating effects of MEG3 on the TDSCs. Finally, experiments in a mouse burn/tenotomy model corroborated the promoting effects of MEG3 on the formation of HO through the miR-129-5p/TCF4/β-catenin axis.Our study demonstrated that the lncRNA MEG3 promoted osteogenic differentiation of TDSCs and thus the formation of heterotopic ossification, which could be a potential therapeutic target.

Biological processes are largely governed by the RNA molecules and resulting peptides that are encoded by an organism’s DNA. For decades, our understanding of biology has been vastly enhanced through study of the distribution and abundance of RNA molecules. Studies of the inner ear are no exception, and approaches like qPCR, RNA-seq, and in situ hybridization (ISH) have contributed greatly to our understanding of inner ear development and function. While qPCR and RNA-seq provide sensitive and broad measures of RNA quantity, they can be limited in their ability to resolve RNA localization. Thus, ISH remains a vital technique for inner ear studies. However, traditional ISH approaches can be technically challenging, time-consuming, suffer from high background, and are generally limited to the investigation of only a single RNA of interest. Recent advances in ISH approaches have overcome many of these limitations allowing for speed, high signal-to-noise, and the ability to perform multiplexed ISH where several transcripts of interest can be visualized in the same tissue or section. One such approach is RNAscope which is a commercially available option that allows for ease of use and, for many transcripts, the ability to achieve absolute quantification of RNA molecules per cell. Here we outline RNAscope methods that have been optimized for inner ear (and related) tissues and allow for relatively rapid labeling of RNA transcripts of interest in fixed tissues. Furthermore, these methods elucidate how RNAscope labeling can be imaged with brightfield or fluorescence microscopy, how it allows for quantification as well as localization, how it can be multiplexed to visualize multiple transcripts simultaneously, and how it can be combined with immunocytochemistry so that RNA and proteins may be visualized in the same sample.

In the developing subpallium, the fate decision between neurons and glia is driven by expression of Dlx1/2 or Olig1/2, respectively, two sets of transcription factors with a mutually repressive relationship. The mechanism by which Dlx1/2 repress progenitor and oligodendrocyte fate, while promoting transcription of genes needed for differentiation, is not fully understood. We identified a motif within DLX1 that binds RBBP4, a NuRD complex subunit. ChIP-seq studies of genomic occupancy of DLX1 and six different members of the NuRD complex show that DLX1 and NuRD colocalize to putative regulatory elements enriched near other transcription factor genes. Loss of Dlx1/2 leads to dysregulation of genome accessibility at putative regulatory elements near genes repressed by Dlx1/2, including Olig2. Consequently, heterozygosity of Dlx1/2 and Rbbp4 leads to an increase in the production of OLIG2+ cells. These findings highlight the importance of the interplay between transcription factors and chromatin remodelers in regulating cell-fate decisions.