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A robust method to isolate Drosophila fat body nuclei for transcriptomic analysis.

ABSTRACT: Gene expression profiles are typically described at the level of the tissue or, often in Drosophila, at the level of the whole organism. Collapsing the gene expression of entire tissues into single measures averages over potentially important heterogeneity among the cells that make up that tissue. The advent of single-cell RNA-sequencing technology (sc-RNAseq) allows transcriptomic evaluation of the individual cells that make up a tissue. However, sc-RNAseq requires a high-quality suspension of viable cells or nuclei, and cell dissociation methods that yield healthy cells and nuclei are still lacking for many important tissues. The insect fat body is a polyfunctional tissue responsible for diverse physiological processes and therefore is an important target for sc-RNAseq. The Drosophila adult fat body consists of fragile cells that are difficult to dissociate while maintaining cell viability. As an alternative, we developed a method to isolate single fat body nuclei for RNA-seq. Our isolation method is largely free of mitochondrial contamination and yields higher capture of transcripts per nucleus compared to other nuclei preparation methods. Our method works well for single-cell nuclei sequencing and can potentially be implemented for bulk RNA-seq.

SUBMITTER: Gupta V

PROVIDER: S-EPMC8500699 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The thalamus is an essential relay station in the cortical-subcortical connections. It is characterized by a complex anatomical architecture composed of numerous small nuclei, which mediate the involvement of the thalamus in a wide range of neurological functions. We present a novel framework for segmenting the thalamic nuclei, which explores the orientation distribution functions (ODFs) from diffusion magnetic resonance images at 3 T. The differentiation of the complex intra-thalamic microstructure is improved by using the spherical harmonic (SH) representation of the ODFs, which provides full angular characterization of the diffusion process in each voxel. The clustering was performed using the k-means algorithm initialized in a data-driven manner. The method was tested on 35 healthy volunteers and our results show a robust, reproducible and accurate segmentation of the thalamus in seven nuclei groups. Six of them closely matched the anatomy and were labeled as anterior, ventral anterior, medio-dorsal, ventral latero-ventral, ventral latero-dorsal and pulvinar, while the seventh cluster included the centro-lateral and the latero-posterior nuclei. Results were evaluated both qualitatively, by comparing the segmented nuclei to the histological atlas of Morel, and quantitatively, by measuring the clusters' extent and the clusters' spatial distribution across subjects and hemispheres. We also showed the robustness of our approach across different sequences and scanners, as well as intra-subject reproducibility of the segmented clusters using additional two scan-rescan datasets. We also observed an overlap between the path of the main long-connection tracts passing through the thalamus and the spatial distribution of the nuclei identified with our clustering algorithm. Our approach, based on SH representations of the ODFs, outperforms the one based on angular differences between the principle diffusion directions, which is considered so far as state-of-the-art method. Our findings show an anatomically reliable segmentation of the main groups of thalamic nuclei that could be of potential use in many clinical applications.

Project description:BackgroundIn Drosophila melanogaster dosage compensation of most X-linked genes is mediated by the male-specific lethal (MSL) complex, which includes MOF. MOF acetylates histone H4 at lysine 16 (H4K16ac). The X-linked Larval serum protein one alpha (Lsp1alpha) gene has long been known to be not dosage compensated. Here we have examined possible explanations for why the Lsp1alpha gene is not dosage compensated.ResultsQuantitative RNase protection analysis showed that the genes flanking Lsp1alpha are expressed equally in males and females and confirmed that Lsp1alpha is not dosage compensated. Unlike control X-linked genes, Lsp1alpha was not enriched for H4K16ac in the third instar larval fat body, the tissue in which the gene is actively expressed. X-linked Lsp1alpha promoter-lacZ reporter transgenes are enriched for H4K16ac in third instar larval fat body. An X-linked reporter gene bracketed by Lsp1alpha flanking regions was dosage compensated. One of the genes flanking Lsp1alpha is expressed in the same tissue. This gene shows a modest enrichment for H4K16ac but only at the part of the gene most distant from Lsp1alpha. Phylogenetic analyses of the sequences of the genomes of 12 Drosophila species shows that Lsp1alpha is only present within the melanogaster subgroup of species.ConclusionLsp1alpha is not modified by the MSL complex but is in a region of the X chromosome that is regulated by the MSL complex. The high activity or tissue-specificity of the Lsp1alpha promoter does not prevent regulation by the MSL complex. The regions flanking Lsp1alpha do not appear to block access by the MSL complex. Lsp1alpha appears to have recently evolved within the melanogaster subgroup of Drosophila species. The most likely explanation for why Lsp1alpha is not dosage compensated is that the gene has not evolved a mechanism to independently recruit the MSL complex, possibly because of its recent evolutionary origin, and because there appears to be a low level of bound MSL complex in a nearby gene that is active in the same tissue.

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A robust method to isolate Drosophila fat body nuclei for transcriptomic analysis.

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