basecalling: CASAVA v1.8.2reads aligned to genome using Bowtie2 v 2.0.6identified reads hitting transcripts using TopHat v 2.0.7 with -G --no novel juncs and Refseq dm3-iGenomes.gtfcalculated transcript fpkm values and gene fpkm values using Cufflinks version 2.02 -G with dm3-iGenomes.gtfGenome_build: Refseq annotation file:dm3 and dm3-iGenomes.gtfSupplementary_files_format_and_content: Cufflinks output file: genes.fpkm_tracking:

basecalling: CASAVA v1.8.2reads aligned to genome using Bowtie2 v 2.0.6identified reads hitting transcripts using TopHat v 2.0.7 with -G --no novel juncs and Refseq dm3-iGenomes.gtfcalculated transcript fpkm values and gene fpkm values using Cufflinks version 2.02 -G with dm3-iGenomes.gtfGenome_build: Refseq annotation file: dm3 (Release 5) and dm3-iGenomes.gtfSupplementary_files_format_and_content: Cufflinks output file (.xlsx)

basecalling: CASAVA v1.8.2reads aligned to genome using Bowtie2 v 2.0.6identified reads hitting transcripts using TopHat v 2.0.7 with -G --no novel juncs and Refseq dm3-iGenomes.gtfcalculated transcript fpkm values and gene fpkm values using Cufflinks version 2.02 -G with dm3-iGenomes.gtfGenome_build: Refseq annotation file: dm3 (Release 5) and dm3-iGenomes.gtfSupplementary_files_format_and_content: Cufflinks output file (.xlsx)

basecalling: CASAVA v1.8.2reads aligned to genome using Bowtie2 v 2.0.6identified reads hitting transcripts using TopHat v 2.0.7 with -G --no novel juncs and Refseq dm3-iGenomes.gtfcalculated transcript fpkm values and gene fpkm values using Cufflinks version 2.02 -G with dm3-iGenomes.gtfGenome_build: Refseq annotation file:dm3 and dm3-iGenomes.gtfSupplementary_files_format_and_content: Cufflinks output file: genes.fpkm_tracking:

We generated a library of 1000 Drosophila stocks in which we inserted a construct in the intron of genes allowing expression of GAL4 under control of endogenous promoters while arresting transcription with a polyadenylation signal 3' of the GAL4. This allows numerous applications. First, similar to 90% of insertions in essential genes cause a severe loss-of-function phenotype, an effective way to mutagenize genes. Interestingly, 12/14 chromosomes engineered through CRISPR do not carry second-site lethal mutations. Second, 26/36 (70%) of lethal insertions tested are rescued with a single UAS-cDNA construct. Third, loss-of-function phenotypes associated with many GAL4 insertions can be reverted by excision with UAS-flippase. Fourth, GAL4 driven UAS-GFP/RFP reports tissue and cell-type specificity of gene expression with high sensitivity. We report the expression of hundreds of genes not previously reported. Finally, inserted cassettes can be replaced with GFP or any DNA. These stocks comprise a powerful resource for assessing gene function.

We previously reported a CRISPR-mediated knock-in strategy into introns of Drosophila genes, generating an attP-FRT-SA-T2A-GAL4-polyA-3XP3-EGFP-FRT-attP transgenic library for multiple uses (Lee et al., 2018a). The method relied on double stranded DNA (dsDNA) homology donors with similar to 1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration cassette, followed by enzymatic removal of one strand. Using this method, we generated GFP-tagged proteins that mark organelles in S2 cells. We then describe two dsDNA methods using cheap synthesized donors flanked by 100 nt homology arms and gRNA target sites cloned into a plasmid. Upon injection, donor DNA (1 to 5 kb) is released from the plasmid by Cas9. The cassette integrates efficiently and precisely in vivo. The approach is fast, cheap, and scalable.

Human oocytes frequently generate aneuploid embryos that subsequently miscarry. I n contrast, Drosophila oocytes from outbred laboratory stocks develop fully regardless of maternal age. Since mature Drosophila oocytes are not extensively stored in the ovary under laboratory conditions like they are in the wild, we developed a system to investigate how storage affects oocyte quality. The developmental capacity of stored mature Drosophila oocytes decays in a precise manner over 14 days at 25 degrees C. These oocytes are transcriptionally inactive and persist using ongoing translation of stored mRNAs. Ribosome profiling revealed a progressive 2.3-fold decline in average translational efficiency during storage that correlates with oocyte functional decay. Although normal bipolar meiotic spindles predominate during the first week, oocytes stored for longer periods increasingly show tripolar, monopolar and other spindle defects, and give rise to embryos that fail to develop due to aneuploidy. Thus, meiotic chromosome segregation in mature Drosophila oocytes is uniquely sensitive to prolonged storage. Our work suggests the chromosome instability of human embryos could be mitigated by reducing the period of time mature human oocytes are stored in the ovary prior to ovulation.

Adult Drosophila Malpighian tubules have low rates of cell turnover but are vulnerable to damage caused by stones, like their mammalian counterparts, kidneys. We show that Drosophilarenal stem cells (RSCs) in the ureter and lower tubules comprise a unique, unipotent regenerative compartment. RSCs respond only to loss of nearby principal cells (PCs), cells critical for maintaining ionic balance. Large polyploid PCs are outnumbered by RSCs, which replace each lost cell with multiple PCs of lower ploidy. Notably, RSCs do not replenish principal cells or stellate cells in the upper tubules. RSCs generate daughters by asymmetric Notch signaling, yet RSCs remain quiescent (cell cycle-arrested) without damage. Nevertheless, the capacity for RSC-mediated repair extends the lifespan of flies carrying kidney stones. We propose that abundant, RSC-like stem cells exist in other tissues with low rates of turnover where they may have been mistaken for differentiated tissue cells.

Polycomb silencing represses gene expression and provides a molecular memory of chromatin state that is essential for animal development. We show that Drosophila female germline stem cells (GSCs) provide a powerful system for studying Polycomb silencing. GSCs have a non-canonical distribution of PRC2 activity and lack silenced chromatin like embryonic progenitors. As GSC daughters differentiate into nurse cells and oocytes, nurse cells, like embryonic somatic cells, silence genes in traditional Polycomb domains and in generally inactive chromatin. Developmentally controlled expression of two Polycomb repressive complex 2 (PRC2)-interacting proteins, Pcl and Scm, initiate silencing during differentiation. In GSCs, abundant Pcl inhibits PRC2-dependent silencing globally, while in nurse cells Pcl declines and newly induced Scm concentrates PRC2 activity on traditional Polycomb domains. Our results suggest that PRC2-dependent silencing is developmentally regulated by accessory proteins that either increase the concentration of PRC2 at target sites or inhibit the rate that PRC2 samples chromatin.

We sequenced more than 52,500 single cells from E11.5 to P5 gonads to analyze primordial follicles and wave 1 medullar follicles during mouse fetal and perinatal oogenesis. Germ cells clustered into six meiotic substages as well as dying/nurse cells. We also define genes expressed by epithelial progenitors, and clarify the similar but distinct genetic programs of bipotential-derived and epithelial-derived pre-granulosa cell progenitors. Their differentially expressed genes are candidates to control the distinctive developmental programs of wave 1 and wave 2 follicles. These observations provide a strong basis for further studies of the development, physiology, and evolutionary conservation of mammalian ovarian follicle formation.