Transcriptomes of somatic and germ line cells in the developing larval ovary of Drosophila melanogaster in wild type, hpoRNAi and ykiRNAi conditions.
Adult insect ovaries are made up of egg production structures called ovarioles, whose number is directly linked to fecundity and fitness. Ovariole number is directly determined by the number of cell stacks called terminal filaments in the larval ovary, which in turn is determined by the number of terminal filament cells available to form terminal filaments. Elucidating the developmental and genetic regulation of terminal filament cell proliferation is thus key to understanding the regulation of adult ovariole number and fitness. Our previous work showed that the Hippo signaling pathway, including the kinase hippo and the transcription factor yorkie, regulate terminal filament cell number in Drosophila melanogaster. To identify the specific transcriptional responses of ovarian cells to Hippo signaling during ovary development, we systematically measured mRNA expression in isolated pools of somatic and germ line cells from the Drosophila larval ovary at the beginning, middle and end of terminal filament formation using RNA-seq. We performed these RNA-seq experiments in wild type, hippoRNAi and yorkieRNAi genetic backgrounds at each of these three developmental stages. Overall design: We extracted RNA from Fluorescence Activated Cell Sorted (FACS) GFP-expressing somatic or germ line cells from the larval ovaries of Drosophila melanogaster at three stages - the beginning (72 hours After Egg Laying, h (AEL)), middle (96h AEL) and end (120h AEL) of terminal filament formation. We specifically marked either somatic or germ cells with GFP using somatic- and germ line-specific GAL4 drivers (w[*];P{w[+mW.hs]=GawB}bab1[PGAL4-2]/TM6B,Tb[1], abbreviated herein as bab:GAL4; Bloomington Drosophila Stock Center (BDSC) #6803 and P{w[+mC]=UAS-Dcr-2.D}1,w[1118];P{w[+mC]=GAL4-nos.NGT}40,  abbreviated herein as nos:GAL4; BDSC #25751 respectively). RNA-seq data from FACS-sorted somatic and germ line cells from wild type ovaries were previously reported in Bioproject PRJNA721702. In the present dataset we report RNA-seq data from FACS-sorted cells in loss of function conditions for two members of the hippo signaling pathway. We knocked down either hippo (using hpo:RNAi line BDSC #33614) or yorkie (using yki:RNAi line BDSC #34067) in either germ line or somatic cells at each of the three developmental stages. The targeted cells also expressed GFP, which was used to separate somatic and germ cells using FACS. Total RNA was isolated from each genetic background/stage/cell type in both control and knockdown conditions. In three biological replicates of each unique experimental condition, cDNA libraries were prepared for each sample from Poly-A enriched-RNA using PrepX kit (Takara 640096). These cDNA libraries were sequenced using a single end 50bp protocol in an Illumina HiSeq 2500 sequencer. Sequences obtained were checked for quality and replicates with 10 million or more reads were used for downstream differential expression analysis. Only one replicate each of sufficient quality was obtained for the early-stage germ line knockdown conditions. Data from these samples were not included in the final analysis but are deposited here. Please note that the following control samples from GSE172015 have been re-used: BioProject accession: PRJNA721702 GEO Sample  SRA Runs  BioSample   Title ==========  ========  =========   ===== GSM5239772  SRR14231550,SRR14231551 SAMN18736523  S_ctrl_Early_1 GSM5239773  SRR14231552,SRR14231553 SAMN18736522  S_ctrl_Early_2 GSM5239774  SRR14231554,SRR14231555 SAMN18736521  S_ctrl_Early_3 GSM5239775  SRR14231556,SRR14231557,SRR14231558,SRR14231559 SAMN18736535  S_ctrl_Mid_1 GSM5239776  SRR14231560,SRR14231561 SAMN18736534  S_ctrl_Mid_2 GSM5239777  SRR14231562,SRR14231563 SAMN18736533  S_ctrl_Mid_3 GSM5239778  SRR14231564   SAMN18736532  S_ctrl_Late_1 GSM5239779  SRR14231565   SAMN18736520  S_ctrl_Late_2 GSM5239780  SRR14231566   SAMN18736519  S_ctrl_Late_3 GSM5239781  SRR14231567,SRR14231568 SAMN18736518  G_ctrl_Early_1 GSM5239782  SRR14231569,SRR14231570 SAMN18736517  G_ctrl_Early_2 GSM5239783  SRR14231571,SRR14231572 SAMN18736516  G_ctrl_Early_3 GSM5239784  SRR14231573,SRR14231574 SAMN18736515  G_ctrl_Mid_1 GSM5239785  SRR14231575,SRR14231576,SRR14231577,SRR14231578 SAMN18736514  G_ctrl_Mid_3 GSM5239786  SRR14231579,SRR14231580 SAMN18736513  G_ctrl_Mid_4 GSM5239787  SRR14231581,SRR14231582,SRR14231583,SRR14231584 SAMN18736512  G_ctrl_Late_2 GSM5239788  SRR14231585,SRR14231586 SAMN18736511  G_ctrl_Late_3 GSM5239789  SRR14231587,SRR14231588 SAMN18736510  G_ctrl_Late_4