A single-cell atlas of Drosophila trachea reveals glycosylation-mediated Notch signaling in cell fate specification

The Drosophila tracheal system is a favorable model for investigating the program of tubular morphogenesis. This system is established in the embryo by post-mitotic cells, but also undergoes remodeling by adult stem cells. Here, we provide a comprehensive cell atlas of Drosophila trachea using the single-cell RNA-sequencing (scRNA-seq) technique. The atlas documents transcriptional profiles of tracheoblasts within the Drosophila airway, delineating 9 major subtypes. Further evidence gained from in silico as well as genetic investigations highlight a set of transcription factors characterized by their capacity to switch cell fate. Notably, the transcription factors Pebbled, Blistered, Knirps, Spalt and Cut are influenced by Notch signaling and determine tracheal cell identity. Moreover, Notch signaling orchestrates transcriptional activities essential for tracheoblast differentiation and responds to protein glycosylation that is induced by high sugar diet. Therefore, our study yields a single-cell transcriptomic atlas of tracheal development and regeneration, and suggests a glycosylation-responsive Notch signaling in cell fate determination.

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-The clean reads of SMART-seq data were mapped to the Drosophila genome sequence using Hisat2 with default parameters.
-The number of mapped reads were counted by featureCounts.
-Cellranger was utilized to align reads of scRNA-seq to the reference genome Drosophila melanogaster and to perform batch effect correction and dataset aggregation https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger.
All data generated or analysed during this study are included in this published article (and its supplementary information files).The SMART-Seq data (L3, 0hr APF and 2hr APF) generated and analyzed in this study have been deposited in the NCBI database under accession number GSE184856 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE184856].The single-cell RNA sequencing data and the SMART-Seq data (control and mmyRNAi) generated and analyzed in this study have been deposited in the NCBI database under accession number GSE240777 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE240777].The databases/ datasets used in the study: Drosophila genome (dm6) https://www.ncbi.nlm.nih.gov/assembly/GCF_000001215.4/ The sample size was determined by our preliminary experiment.
-More than 3 white pupae were used for migration experiment, EdU cell proliferation assay, and most immunofluorescence staining.According to our preliminary experiments, 3 is the minimal sample size to obtain convincing and statistically significant conclusions.
-To minimize the sample variation between different individuals, clusters of tracheal progenitors were collected from two L3 or pupae that were at the same developmental stage.
-Our preliminary results revealed that the sample size needs to be larger than 50, which grants sufficient yield for next generation sequencing.
-The size number of GlcNAcylation detection is 150 for each sample.According to our preliminary experiments, smaller sample size may not be sufficient to obtain clear and sharp bands.
-The size number of O-GlcNAc detection is 80 for each sample.our preliminary experiments show that smaller sample size may lead to blurry or even undetectable bands.
-The size number of Immunoprecipitation is 150 for each sample.The sample size was chosen based on our preliminary experiments to obtain enough protein of interest for the following SDS-PAGE. None.
At least 3 independent biological replicates were performed successfully for most experiments.
Animals were allocated to each experimental group randomly for all experiments.
Image qualifications were performed blindly.For most experiments, blinding is not possible during sample collection as genotypes of flies Policy information about availability of of computer codeData collectionHai Huang, Quanyi Zhao, Honggang Wu Wu Feb 16, 2024 -Confocal images were collected using Zen 3.1 (blue edition) software.-Samplesfor SMART-seq was sequenced by by Hiseq4000 instrument.-Samples(L3, 0hr APF and 2hr APF) for SMART-seq was sequenced by by llumina NovaSeq 6000 instrument.-Samples for scRNA-seq was sequenced by by Illumina NovaSeq 6000 instrument.