Serotonergic Neurons in the Chick Brainstem Express Various Serotonin Receptor Subfamily Genes

Serotonin (5-hydroxytryptamine, 5-HT) is a phylogenetically conserved modulatory neurotransmitter. In mammals, 5-HT plays an important role in the regulation of many mental states and the processing of emotions in the central nervous system. Serotonergic neurons in the central nervous system, including the dorsal raphe (DR) and median raphe (MR) nuclei, are spatially clustered in the brainstem and provide ascending innervation to the entire forebrain and midbrain. Both between and within the DR and MR, these serotonergic neurons have different cellular characteristics, developmental origin, connectivity, physiology, and related behavioral functions. Recently, an understanding of the heterogeneity of the DR and MR serotonergic neurons has been developed at the molecular level. In birds, emotion-related behavior is suggested to be modulated by the 5-HT system. However, correspondence between the raphe nuclei of birds and mammals, as well as the cellular heterogeneity in the serotonergic neurons of birds are poorly understood. To further understand the heterogeneity of serotonergic neurons in birds, we performed a molecular dissection of the chick brainstem using in situ hybridization. In this study, we prepared RNA probes for chick orthologs of the following serotonin receptor genes: 5-HTR1A, 5-HTR1B, 5-HTR1D, 5-HTR1E, 5-HTR1F, 5-HTR2A, 5-HTR2B, 5-HTR2C, 5-HTR3A, 5-HTR4, 5-HTR5A, and 5-HTR7. We showed that the expression pattern of 5-HT receptors in the serotonin neurons of chick DR and MR may vary, suggesting heterogeneity among and within the serotonin neurons of the DR and MR in the chick brainstem. Our findings regarding the molecular properties of serotonergic neurons in the bird raphe system will facilitate a good understanding of the correspondence between bird and mammalian raphes.

Quantitative intracellular retention of delivered RNAs through optimized cell fixation and immuno-staining.

Detection of nucleic acids within sub-cellular compartments is key to understanding their function. Determining the intracellular distribution of nucleic acids requires quantitative retention and estimation of their association with different organelles by immunofluorescence microscopy. This is particularly important for the delivery of nucleic acid therapeutics which depends on endocytic uptake and endosomal escape. However, the current protocols fail to preserve the majority of exogenously delivered nucleic acids in the cytoplasm. To solve this problem, by monitoring Cy5-labeled mRNA delivered to primary human adipocytes via lipid nanoparticles (LNP), we optimized cell fixation, permeabilization and immuno-staining of a number of organelle markers, achieving quantitative retention of mRNA and allowing visualization of levels which escape detection using conventional procedures. The optimized protocol proved effective on exogenously delivered siRNA, miRNA, as well as endogenous miRNA. Our protocol is compatible with RNA probes of single molecule fluorescence in-situ hybridization (smFISH) and molecular beacon, thus demonstrating that it is broadly applicable to study a variety of nucleic acids in cultured cells.

Small RNAs Asserting Big Roles in Mycobacteria

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), with 10.4 million new cases per year reported in the human population. Recent studies on the Mtb transcriptome have revealed the abundance of noncoding RNAs expressed at various phases of mycobacteria growth, in culture, in infected mammalian cells and in patients. Among these noncoding RNAs are both small RNAs (sRNAs) between 50-350 nts in length and smaller RNAs (sncRNA) <50 nts. In this review, we provide an up-to-date synopsis of the identification, designation, and function of these Mtb-encoded sRNAs and sncRNAs. The methodological advances including RNA sequencing strategies, small RNA antagonists and locked nucleic acid sequence specific RNA probes advancing the studies on these small RNA are described. Initial insights into the regulation of the small RNA expression and putative processing enzymes required for their synthesis and function are discussed. There are many open questions remaining about the biological and pathogenic roles of these small non-coding RNAs, and potential research directions needed to define the role of these mycobacterial noncoding RNAs summarized.

hyRAD RNA probes preparation and capture v2

Supplemental Information for: Suchan, T., Kusliy, M.A., Khan, N., Chauvey, L., Tonasso-Calvière, L., Schiavinato, S., Southon, J., Keller, M., Kitagawa, K., Krause, J., Bessudnov, A.N., Bessudnov, A.A., Graphodatsky, A.S., Lamas, S.V., Wilczyński, J., Pospuła, S., Tunia, K., Nowak, M., Moskal-delHoyo, M., Tishkin, A.A., Pryor, A.J.E., Outram, A.K., Orlando, L. (2021) Performance and automation of ancient DNA capture with RNA hyRAD probes. Molecular Ecology Resources, doi: 10.1111/1755-0998.13518

Identification of the cell-type-specific ER membrane protein Tanmp expressed in hypothalamic tanycytes and subsets of neurons

Genomes of higher eukaryotes encode many uncharacterized proteins, and the functions of these proteins cannot be predicted from the primary sequences due to a lack of conserved functional domains. During a screening of novel noncoding RNAs abundantly expressed in mouse brains, we incidentally identified a gene termed Tanmp, which encoded an endoplasmic reticulum (ER) protein without known functional domains. Tanmp is specifically expressed in the nervous system, and the highest expression was observed in a specialized cell type called tanycyte that aligns the ventral wall of the third ventricle in the hypothalamus. Immunostaining of Tanmp revealed the fine morphology of tanycytes with highly branched apical ER membranes. Immunoprecipitation revealed that Tanmp associates with mitochondrial ATPase at least in vitro, and ER and mitochondrial signals occasionally overlapped in tanycytes. Mutant mice lacking Tanmp did not exhibit overt phenotypes, suggesting that Tanmp is not essential in mice reared under normal laboratory conditions. We also found that RNA probes that are predicted to uniquely detect Tanmp mRNA cross-reacted with uncharacterized RNAs, highlighting the importance of experimental validation of the specificity of probes during the hybridization-based study of RNA localization.

Quantitative intracellular retention of delivered RNAs through optimized cell fixation and immuno-staining

Detection of nucleic acids within sub-cellular compartments is key to understanding their function. Determining the intracellular distribution of nucleic acids requires quantitative retention and estimation of their association with different organelles by immunofluorescence microscopy. This is important also for the delivery of nucleic acid therapeutics which depends on endocytic uptake and endosomal escape. However, the current methods fail to preserve the majority of exogenously delivered nucleic acids in the cytoplasm. To solve this problem, by monitoring Cy5-labeled mRNA delivered to primary human adipocytes via lipid nanoparticles (LNP), we optimized cell fixation, permeabilization and immuno-staining of a number of organelle markers, achieving quantitative retention of mRNA and allowing visualization of levels which escape detection using conventional procedures. Additionally, we demonstrated the protocol to be effective on exogenously delivered siRNA, miRNA, as well as endogenous miRNA. Our protocol is compatible with RNA probes of single molecule fluorescence in-situ hybridization (smFISH) and molecular beacon, thus demonstrating that it is broadly applicable to study a variety of nucleic acids.

Alternative Promoter Use Governs the Expression of IgLONCell Adhesion Moleculesin Histogenetic Fields of the Embryonic Mouse Brain

The members of the IgLON superfamily of cell adhesion molecules facilitate fundamental cellular communication during brain development, maintain functional brain circuitry, and are associated with several neuropsychiatric disorders such as depression, autism, schizophrenia, and intellectual disabilities. Usage of alternative promoter-specific 1a and 1b mRNA isoforms in Lsamp, Opcml, Ntm, and the single promoter of Negr1 in the mouse and human brain has been previously described. To determine the precise spatiotemporal expression dynamics of Lsamp, Opcml, Ntm isoforms, and Negr1, in the developing brain, we generated isoform-specific RNA probes and carried out in situ hybridization in the developing (embryonic, E10.5, E11.5, 13.5, 17; postnatal, P0) and adult mouse brains. We show that promoter-specific expression of IgLONs is established early during pallial development (at E10.5), where it remains throughout its differentiation through adulthood. In the diencephalon, midbrain, and hindbrain, strong expression patterns are initiated a few days later and begin fading after birth, being only faintly expressed during adulthood. Thus, the expression of specific IgLONs in the developing brain may provide the means for regionally specific functionality as well as for specific regional vulnerabilities. The current study will therefore improve the understanding of how IgLON genes are implicated in the development of neuropsychiatric disorders.

Continuous Polony Gels for Tissue Mapping with High Resolution and RNA Capture Efficiency

Current technologies for acquiring spatial transcript information from tissue sections rely on either RNA probes or spatial barcodes. The former methods require a priori knowledge for probeset formulation; the latter have yet to achieve single cell resolution and/or transcript capture efficiencies approaching dissociative, single-cell methods. Here, we describe a novel spatial transcriptome assay called polony (or DNA cluster)-indexed library-sequencing (PIXEL-seq). It improves upon other spatial barcoding methods by employing “continuous” polony oligos arrayed across a customized gel surface. In terms of assay performance, PIXEL-seq attains ≤ 1 µm resolution and captures >1,000 unique molecular identifiers/10×10 µm2. In other words, this global, naive platform achieves subcellular spatial transcriptome mapping while maintaining high transcript capture efficiencies.