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2 results on '"Wiegleb, Gordon"'

1. Tissue dissociation for single-cell and single-nuclei RNA sequencing for low amounts of input material.

Author

Wiegleb, Gordon, Reinhardt, Susanne, Dahl, Andreas, and Posnien, Nico

Subjects
*IMAGINAL disks, *DROSOPHILA melanogaster, *SYNTHETIC genes, *GENE expression, *TISSUES
Abstract

Background: Recent technological advances opened the opportunity to simultaneously study gene expression for thousands of individual cells on a genome-wide scale. The experimental accessibility of such single-cell RNA sequencing (scRNAseq) approaches allowed gaining insights into the cell type composition of heterogeneous tissue samples of animal model systems and emerging models alike. A major prerequisite for a successful application of the method is the dissociation of complex tissues into individual cells, which often requires large amounts of input material and harsh mechanical, chemical and temperature conditions. However, the availability of tissue material may be limited for small animals, specific organs, certain developmental stages or if samples need to be acquired from collected specimens. Therefore, we evaluated different dissociation protocols to obtain single cells from small tissue samples of Drosophila melanogaster eye-antennal imaginal discs. Results: We show that a combination of mechanical and chemical dissociation resulted in sufficient high-quality cells. As an alternative, we tested protocols for the isolation of single nuclei, which turned out to be highly efficient for fresh and frozen tissue samples. Eventually, we performed scRNAseq and single-nuclei RNA sequencing (snRNAseq) to show that the best protocols for both methods successfully identified relevant cell types. At the same time, snRNAseq resulted in less artificial gene expression that is caused by rather harsh dissociation conditions needed to obtain single cells for scRNAseq. A direct comparison of scRNAseq and snRNAseq data revealed that both datasets share biologically relevant genes among the most variable genes, and we showed differences in the relative contribution of the two approaches to identified cell types. Conclusion: We present two dissociation protocols that allow isolating single cells and single nuclei, respectively, from low input material. Both protocols resulted in extraction of high-quality RNA for subsequent scRNAseq or snRNAseq applications. If tissue availability is limited, we recommend the snRNAseq procedure of fresh or frozen tissue samples as it is perfectly suited to obtain thorough insights into cellular diversity of complex tissue. [ABSTRACT FROM AUTHOR]

Published
2022
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2. Dynamic genome wide expression profiling of Drosophila head development reveals a novel role of Hunchback in retinal glia cell development and blood-brain barrier integrity

Author

Torres-Oliva, Montserrat, Schneider, Julia, Wiegleb, Gordon, Kaufholz, Felix, and Posnien, Nico

Subjects
Central Nervous System, Embryo, Nonmammalian, lcsh:QH426-470, Arthropoda, Organogenesis, Gene Expression, Research and Analysis Methods, Biochemistry, Nervous System, Retina, Polyploidy, Animals, Genetically Modified, Model Organisms, Ocular System, DNA-binding proteins, Genetics, Medicine and Health Sciences, Animals, Drosophila Proteins, Gene Regulation, Drosophila Melanogaster, Transcriptional Control, Gene Expression Profiling, Organisms, Biology and Life Sciences, Proteins, Eukaryota, Gene Expression Regulation, Developmental, Cell Differentiation, Animal Models, Invertebrates, Regulatory Proteins, Insects, lcsh:Genetics, Experimental Organism Systems, Blood-Brain Barrier, Eyes, Drosophila, Anatomy, Head, Departures from Diploidy, Neuroglia, Research Article, Transcription Factors
Abstract

Drosophila melanogaster head development represents a valuable process to study the developmental control of various organs, such as the antennae, the dorsal ocelli and the compound eyes from a common precursor, the eye-antennal imaginal disc. While the gene regulatory network underlying compound eye development has been extensively studied, the key transcription factors regulating the formation of other head structures from the same imaginal disc are largely unknown. We obtained the developmental transcriptome of the eye-antennal discs covering late patterning processes at the late 2nd larval instar stage to the onset and progression of differentiation at the end of larval development. We revealed the expression profiles of all genes expressed during eye-antennal disc development and we determined temporally co-expressed genes by hierarchical clustering. Since co-expressed genes may be regulated by common transcriptional regulators, we combined our transcriptome dataset with publicly available ChIP-seq data to identify central transcription factors that co-regulate genes during head development. Besides the identification of already known and well-described transcription factors, we show that the transcription factor Hunchback (Hb) regulates a significant number of genes that are expressed during late differentiation stages. We confirm that hb is expressed in two polyploid subperineurial glia cells (carpet cells) and a thorough functional analysis shows that loss of Hb function results in a loss of carpet cells in the eye-antennal disc. Additionally, we provide for the first time functional data indicating that carpet cells are an integral part of the blood-brain barrier. Eventually, we combined our expression data with a de novo Hb motif search to reveal stage specific putative target genes of which we find a significant number indeed expressed in carpet cells., Author summary The development of different cell types must be tightly coordinated, and the eye-antennal imaginal discs of Drosophila melanogaster represent an excellent model to study the molecular mechanisms underlying this coordination. These imaginal discs contain the anlagen of nearly all adult head structures, such as the antennae, the head cuticle, the ocelli and the compound eyes. While large scale screens have been performed to unravel the gene regulatory network underlying compound eye development, a comprehensive understanding of genome wide expression dynamics throughout head development is still missing to date. We studied the genome wide gene expression dynamics during eye-antennal disc development in D. melanogaster to identify new central regulators of the underlying gene regulatory network. Expression based gene clustering and transcription factor motif enrichment analyses revealed a central regulatory role of the transcription factor Hunchback (Hb). We confirmed that hb is expressed in two polyploid retinal subperineurial glia cells (carpet cells). Our functional analysis shows that Hb is necessary for carpet cell development and we show for the first time that the carpet cells are an integral part of the blood-brain barrier.

Published
2018

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