Methods to Study the Myenteric Plexus of Rat Small Intestine

The close interaction between the enteric nervous system, microbiome, and brain in vertebrates is an emerging topic of recent studies. Different species such as rat, mouse, and human are currently being used for this purpose, among others. The transferability of protocols for tissue isolation and sample collection is not always straightforward. Thus, the present work presents a new protocol for isolation and sample collection of rat myenteric plexus cells for in vivo as well as in vitro studies. With the methods and chemicals described in detail, a wide variety of investigations can be performed with regard to normal physiological as well as pathological processes in the postnatal developing enteric nervous system. The fast and efficient preparation of the intestine as the first step is particularly important. We have developed and described a LIENS chamber to obtain optimal tissue quality during intestinal freezing. Cryosections of the flat, snap-frozen intestine can then be prepared for histological examination of the various wall layers of the intestine, e.g. by immunohistochemistry. In addition, these cryosections are suitable for the preparation of defined regions, as shown here using the ganglia of the mesenteric plexus. This specific tissue was obtained by laser microdissection, making the presented methodology also suitable for subsequent analyses that require high quality (specificity) of the samples. Furthermore, we present here a fully modernized protocol for the cultivation of myenteric neurons from the rat intestine, which is suitable for a variety of in vitro studies.

Laser Microdissection of Pisum sativum L. Nodules Followed by RNA-Seq Analysis Revealed Crucial Transcriptomic Changes During Infected Cell Differentiation

Garden pea (Pisum sativum L.) is a globally important legume crop. Like other legumes, it forms beneficial symbiotic interactions with the soil bacteria rhizobia, gaining the ability to fix atmospheric nitrogen. In pea nodules, the meristem is long-lasting and results in the formation of several histological zones that implicate a notable differentiation of infected host cells. However, the fine transcriptional changes that accompany differentiation are still unknown. In this study, using laser microdissection followed by RNA-seq analysis, we performed transcriptomic profiling in the early infection zone, late infection zone, and nitrogen fixation zone of 11-day-old nodules of pea wild-type line SGE. As a result, a list of functional groups of differentially expressed genes (DEGs) in different nodule histological zones and a list of genes with the most prominent expression changes during nodule development were obtained. Their analyses demonstrated that the highest amount of DEGs was associated with the nitrogen fixation zone. Among well-known genes controlling nodule development, we revealed genes that can be novel players throughout nodule formation. The characterized genes in pea were compared with those previously described in other legumes and their possible functions in nodule development are discussed.

Olig2-astrocytes express neutral amino acid transporter SLC7A10 (Asc-1) in the adult brain

We have reported that the transcription factor Olig2 labels a subpopulation of astrocytes (Olig2-astrocytes), which show distribution patterns different from those of GFAP-expressing astrocytes (GFAP-astrocytes) in the adult brain. Here, to uncover the specific functions of Olig2-astrocytes, we first analyzed public single-cell RNA-seq databases of adult mouse brains. Unbiased classification of gene expression profiles and subsequent gene ontology analyses revealed that the majority of Olig2-astrocytes belonged to an astrocytic cluster that is enriched for transporter-related genes. SLC7A10 (also known as ASC-1) was one of the representative neutral amino acid transporter genes in the cluster. To complement the in silico data analyses, we differentially isolated Olig2- and GFAP-astrocytes from the same frozen section of the lateral globus pallidus using laser microdissection and compared their gene expression by quantitative reverse transcription PCR. We confirmed that Olig2 and GFAP mRNAs were preferentially expressed in the Olig2- and GFAP-astrocytes, respectively, indicating that the laser microdissection method yielded minimal cross-contamination between two types of cells. The Olig2-astrocytes expressed significantly higher levels of SLC7A10 mRNA than the GFAP-astrocytes, corroborating the in silico data. We next localized SLC7A10 protein by immunohistochemistry in the lateral globus pallidus, which was also genetically labeled for Olig2. SLC7A10 co-localized with Olig2-genetic labeling, especially on the fine processes of Olig2-astrocytes. These results are consistent with the recent discovery that SLC7A10 is expressed not only in neurons but also in a subset of astrocytes. Taken together, our findings suggest that SLC7A10 exerts specific functions in Olig2-astrocytes of the adult brain.

Laser Microdissection Transcriptome Data Derived Gene Regulatory Networks of Developing Rice Endosperm Revealed Tissue- and Stage-specific Regulators Modulating Starch Metabolism

Rice (Oryza sativa L.) filial seed tissues are heterozygous in its function, which accumulate distinct storage compounds spatially in starchy endosperm and aleurone. In this study, we identified the 18 tissue- and stage-specific gene co-regulons in the developing endosperm by isolating four fine tissues dorsal aleurone layer (AL), central starchy endosperm (CSE), dorsal starchy endosperm (DSE), and lateral starchy endosperm (LSE) at two developmental stages (7 days after flowering, DAF and 12DAF) using laser microdissection (LM) coupled with gene expression analysis of a 44K microarray. The derived co-expression regulatory networks depict that distinct set of starch biosynthesis genes expressed preferentially at first in CSE at 7 DAF and extend its spatial expression to LSE and DSE by 12 DAF. Interestingly, along with the peak of starch metabolism we noticed accumulation of transcripts related to phospholipid and glycolipid metabolism in CSE during 12 DAF. The spatial distribution of starch accumulation in distinct zones of starchy endosperm contains specific transcriptional factors and hormonal-regulated genes. Genes related to programmed cell death (PCD) were specifically expressed in CSE at 12DAF, when starch accumulation was already completed in that tissue. The aleurone layer present in the outermost endosperm accumulates transcripts of lipid, tricarboxylic acid metabolism, several transporters, while starch metabolism and PCD is not pronounced. These regulatory cascades are likely to play a critical role in determining the positional fate of cells and offer novel insights into the molecular physiological mechanisms of endosperm development from early to middle storage phase.

Erratum: Velada et al. Laser Microdissection of Specific Stem-Base Tissue Types from Olive Microcuttings for Isolation of High-Quality RNA. Biology 2021, 10, 209

The author wishes to make an erratum to the published version of the paper [...]