A gene expression atlas of embryonic neurogenesis in Drosophila reveals complex spatiotemporal regulation of lncRNAs

ABSTRACT Cell type specification during early nervous system development in Drosophila melanogaster requires precise regulation of gene expression in time and space. Resolving the programs driving neurogenesis has been a major challenge owing to the complexity and rapidity with which distinct cell populations arise. To resolve the cell type-specific gene expression dynamics in early nervous system development, we have sequenced the transcriptomes of purified neurogenic cell types across consecutive time points covering crucial events in neurogenesis. The resulting gene expression atlas comprises a detailed resource of global transcriptome dynamics that permits systematic analysis of how cells in the nervous system acquire distinct fates. We resolve known gene expression dynamics and uncover novel expression signatures for hundreds of genes among diverse neurogenic cell types, most of which remain unstudied. We also identified a set of conserved long noncoding RNAs (lncRNAs) that are regulated in a tissue-specific manner and exhibit spatiotemporal expression during neurogenesis with exquisite specificity. lncRNA expression is highly dynamic and demarcates specific subpopulations within neurogenic cell types. Our spatiotemporal transcriptome atlas provides a comprehensive resource for investigating the function of coding genes and noncoding RNAs during crucial stages of early neurogenesis.


Supplementary Materials and Methods
Fly strains and husbandry Drosophila  The IC-GFP and IC-dsRed lines were created by standard P-element transgenesis (Rubin & Spradling 1982) in a y 1 w 1118 background. The ind enhancer (ind_1.4) (Markstein et al. 2004) was cloned using primers (ngctagcgtcgacGCTTCAAAGCTCCGGGAAACG & nctcgagTCTGGGCCTTCGGTCCGAAAATG) flanked with NheI and SalI restriction sites (F primer) and with XhoI (R primer). PCR product was T/A cloned into pCRII-Duo and concatemerized using the compatibly cohesive sites XhoI and SalI. 3x ind_1.4 constructs were directionally subcloned into the P-element vectors pH-Stinger or pRed-HStinger (Barolo et al. 2004) using NheI and XhoI.
Fly stocks were maintained at 25°C, ~60% relative humidity on standard fly food with 12 hr light/dark cycles according to standard procedures. 2-hr embryo collections were done on apple juice agar plates with yeast paste after 3 x 1hr pre-lays in the morning, embryos were then aged for an appropriate amount of time at 25°C and ~60% relative humidity before dechorionation.

DIV-SortSeq
This protocol incorporates portions from MARIS (Hrvatin et al. 2014). All steps after dechorionation were performed on ice, using ice-cold DEPC-treated solutions. Primary and secondary antibodies used in this study are listed in Table S1. RNase-free BSA was obtained from Gemini Bioproducts and is critical for isolation of high-quality RNA.
FACS-purified cells were pelleted and resuspended in 200µl Digestion Buffer (5M NaCl, 1M Tris-HCl pH 8.0, 200mM EDTA, 10% SDS, 3.2U Proteinase K, 1:100 RNase inhibitor) and incubated at 50°C, 15min (Proteinase K digestion) followed by 80°C, 15min (reversal of formaldehyde cross-links). Samples were transferred to ice and resuspended in 600µl TRIzol LS Reagent (Thermo, 10296028). RNA was isolated using the DirectZol RNA MicroPrep Kit (Zymo Research, R2060) according to the manufacturer's instructions. RNA concentration was measured using the Qubit RNA HS Assay kit (Thermo, Q32852) and RNA quality determined using the Agilent RNA 6000 Pico Kit (Agilent, 5067-1513). All RNA-seq libraries were constructed using the NuGEN Ovation Drosophila RNA-Seq System with 10 ng -100 ng total RNA input. Library concentration was quantified using the Qubit dsDNA HS Assay (Thermo, Q32854) and quality was determined on a BioAnalyzer TM using Agilent High Sensitivity DNA Kits (Agilent, 5067-4626). All libraries were sequenced on the Illumina HiSeq4000 at a mean depth of 62.5 million 75bp paired-end reads per sample. RNA-seq datasets generated for this study are detailed in Tables S5 and   S6. A detailed, step-wise protocol is available upon request.

Nuclear-cytoplasmic fractionation
The cell fractionation procedure incorporates portions from MARIS (Hrvatin et al. 2014). All steps were performed on ice, using ice-cold DEPC-treated solutions, and all centrifugation steps were performed at 4˚C. Embryos were processed to single-cell suspension as described above for DIV-SortSeq, then pelleted and resuspended in Cyto Extract Buffer (20mM Tris pH 7.6, 0.1mM EDTA, 2mM MgCl2). After hypotonic swelling, cells were gently lysed by addition of 0.6% CHAPS for isolation of t he cytoplasmic fraction. Nuclei were pelleted at 500xg for 5min, and the supernatant retained, and an appropriate volume of TRIzol LS Reagent was added (cytoplasmic fraction).
Quantitative RT-PCR (qPCR) 50ng of total RNA was reverse-transcribed with the QuantiTect Reverse Transcription kit (Qiagen, 205310) in a total volume of 20µl, according to the manufacturer's instructions. For each gene, 0.2µl Development: doi:10.1242/dev.175265: Supplementary information cDNA was used for input into qPCR using SensiFAST™ SYBR® No-ROX Kit (Bioline, 98020) and 5µM forward and reverse primers in a total volume of 20µl. qPCR primer sequences are listed in Table S2. qPCR thermal cycling and fluorescent data acquisition was performed using the BioRad CFX96 Touch Real-Time PCR Detection System. Expression fold changes were calculated via the ∆∆CT method (Vandesompele et al. 2002;Schmittgen & Livak 2008), normalized to the mean CT of two reference genes: α-tubulin and actin 42A.
RNA probe design and synthesis for RNA in situ hybridization.
Where available, cDNA clones were obtained from the Drosophila Gene Collection or Drosophila Genomics Resource Center (Stapleton et al. 2002), detailed in Table S3. Constructs were linearized via restriction digestion, and subjected to in vitro transcription using appropriate RNA Polymerases (Roche), using ribonucleotide mixtures containing dUTP-DIG, -FITC, or -Biotin (Roche).
Where cDNA clones were not available, PCR primers were designed to amplify a region within the transcribed locus from genomic DNA. Primer sequences are detailed in Table S4. A T7 promoter sequence appended to the reverse primer allowed in vitro transcription directly from the PCR product.