HO1, NQO1, and GCLC). Functional studies demonstrated that PGK1 shRNA largely attenuated DEX-induced oxidative injury and following death of OB-6 cells and primary osteoblasts. Furthermore, PGK1 knockout, by the CRISPR/Cas9 method, similarly induced Nrf2 signaling activation and protected osteoblasts from DEX. Importantly, PGK1 depletion-induced osteoblast cytoprotection against DEX was almost abolished by Nrf2 shRNA. In addition, Keap1 shRNA mimicked and nullified PGK1 shRNA-induced anti-DEX osteoblast cytoprotection. At last we show that PGK1 expression is downregulated in human necrotic femoral head tissues of DEX-taking patients, correlating with HO1 depletion. Collectively, these results show that PGK1 depletion protects human osteoblasts from DEX via activation of Keap1-Nrf2 signaling cascade.Activation of nuclear-factor-E2-related factor 2 (Nrf2) cascade can alleviate dexamethasone (DEX)-induced oxidative injury and death of human osteoblasts. A recent study has shown that phosphoglycerate kinase 1 (PGK1) inhibition/depletion will lead to Kelch-like ECH-associated protein 1 (Keap1) methylglyoxal modification, thereby activating Nrf2 signaling cascade. Here, in OB-6 osteoblastic cells and primary human osteoblasts, PGK1 silencing, by targeted shRNA, induced Nrf2 signaling cascade activation, causing Nrf2 protein stabilization and nuclear translocation, as well as increased expression of ARE-dependent genes ( Yet, over-dose and/or sustained DEX administration shall induce profound cytotoxic effects to human osteoblasts, leading to osteoporosis, osteonecrosis, or even non-traumatic bone fractures3. In vitro studies have demonstrated that to osteoblasts or osteoblastic cells, DEX treatment will exert direct and profound cytotoxicity, leading to subsequent cell apoptosis and necrosis7. By exploring the pathological mechanisms of DEX-induced osteoblast cell death, recent studies have been testing the novel osteoblast-protective strategies7.Patients suffering the chronic inflammatory and auto-immune diseases are routinely prescribed with glucocorticoids  cascade is a vital and endogenous defensive mechanism against oxidative stress10. Without stimulation, Nrf2 binds to Kelch-like ECH-associated protein 1 (Keap1), subjects to ubiquitination and proteasomal degradation via Cullin 3 (Cul3) E3 ubiquitin ligase10. Activated Nrf2 protein disassociates with Keap1, being stabilized and accumulated in cytosol, and eventually translocates to cell nuclei, where it binds to ARE (antioxidant response element) loci to promote transcription and expression of key cytoprotective and antioxidant genes10. These genes, among others, include heme oxygenase-1 (HO1), NAD(P)H quinone oxidoreductase 1 (NQO1), and γ-glutamyl cysteine ligase catalytic subunit , showing potent antioxidant activity and ability in different human cells10. Furthermore, agents or stimuli that can modify Keap1’s cysteine residues can also induce Keap1-Nrf2 disassociation and Nrf2 cascade activation13.DEX treatment in osteoblasts/osteoblastic cells will lead to reactive oxygen species (ROS) production and profound oxidative injury, causing cell death and apoptosis8. Liu et al. show that activation of the EGFR-Akt-Nrf2 signaling cascade by icariside II protected osteoblasts from DEX14. Compound 991, a novel AMP-activated protein kinase (AMPK) activator, provoked AMPK-dependent Nrf2 signaling to protect osteoblasts from DEX15. MIND4-17, by uniquely separating Nrf2-Keap1 complex, attenuated hydrogen peroxide (H2O2)-induced oxidative stress in osteoblasts15. These results show that activation of Nrf2 cascade by pharmacological strategies protects osteoblasts from DEX and other oxidative stress.Studies have shown that forced activation of Nrf2 cascade shall protect osteoblastic cells/osteoblasts from DEX and other oxidative stresses. For example, Li et al. demonstrated that SC79, a first-in-class Akt activator, protected osteoblasts from DEX via activation of Akt downstream Nrf2 cascade6. By targeting tuberous sclerosis complex 1 (TSC1), microRNA-19a (miR-19a) activated mTOR-dependent Nrf2 cascade to alleviate DEX-induced oxidative injury in osteoblasts7. In addition, microRNA-455 (miR-445) activated Nrf2 signaling and protected osteoblasts from oxidative stress by targeting Cullin 3, the Nrf2’s E3 ligase16. Thus, activation of the Nrf2 cascade, using pharmacological agents or genetic strategies, will exert potent osteoblast cytoprotective actions against DEX-induced oxidative injury.Recent literatures have also been using genetic strategies to activate Nrf2 signaling in human osteoblasts. Zhao et al., demonstrated that microRNA-200a (miR-200a) targeted and silenced Keap1, thus activating Nrf2 signaling and protecting osteoblasts from DEX18. PGK1 catalyzes the reversible conversion of 1,3-diphosphoglycerate and ADP to 3-phosphoglycerate and ATP18. A very recent and interesting study has discovered a key role of PGK1 in shutting down Nrf2 cascade activation19. PGK1 inhibition or depletion will lead to the accumulation of the reactive metabolite methylglyoxal to modify Keap119. The latter will then form a characteristic methylimidazole crosslink between proximal cysteine and arginine residues (MICA), causing Keap1 dimerization, followed by Keap1-Nrf2 disassociation and activation of Nrf2 cascade19. PKG1 expression and potential functions in human osteoblasts have not been studied thus far. In the present study we will show that PGK1 depletion activates Nrf2 signaling to protect human osteoblasts from DEX.In the glycolytic pathway, phosphoglycerate kinase 1 (PGK1) is an essential enzyme for ATP generationDEX, polybrene, puromycin, JC-1 dye, MG-132, cycloheximide (CHX), and 3--2,5 diphenyltetrazolium bromide (MTT) dye were obtained from Sigma-Aldrich Chemicals . The following antibodies for p44/42 MAPK , HO1 (#70081), NQO1 (#3187), Nrf2 (#12721), Keap1 (#8047), α-Tubulin (#2125), and Lamin B1 (#13435) as well as cleaved-poly (ADP-ribose) polymerase , cleaved-caspase-3 (#9664) were purchased from Cell Signaling Tech . The anti-GCLC antibody (ab55435) and the anti-adenine nucleotide translocase 1 (ANT-1) antibody (ab102032) were provided by Abcam . The following antibodies for PGK1 (sc-130335), cyclophilin-D , and VDAC1 (sc-390996) were purchased from Santa Cruz Biotech Co. . The reagents for cell culturing, including fetal bovine serum (FBS), DMEM, penicillin and streptomycin were obtained from Gibco-BRL Co. . TRIzol and other RNA assay agents, Annexin V, propidium iodide (PI) and cell transfection reagents (Lipofectamine 2000 and others) were from Invitrogen Thermo-Fisher . mRNA primers were purchased from Genechem Co. . All viral constructs and sequences were also designed and provided by Genechem Co.2 human osteoblastic cells, the primary human osteoblasts  as well as HEK-293T cells were described early20. Cells were subjected to mycoplasma and microbial contamination examination every 3 months. Authentication by STR profiling, population doubling time, and morphology were checked as well to confirm the genotype. Primary human osteoblasts were utilized at passages 3–10. The protocols using the primary human cells were approved by the Ethics Committee of all authors institutions, according to the principles of Declaration of Helsinki.Cultures of the established OB-621, with qPCR performed by a SYBR Green I real-time PCR kit 22. The primers for qPCR assays were listed in Table GAPDH.Following the applied treatment, total RNA was extracted and reverse transcribed as described in ref. 23. The ImageJ software  was utilized for the quantification of targeted protein band, with its value normalized to the loading control.The detailed procedures of western blotting were described elsewhere24.The protocol of Mito-IP, examining the mitochondrial cyclophilin-D (CyPD)-adenine nucleotide translocator 1 (ANT-1) association, the indicator of programmed necrosis pathway activation, was described elsewherehuman PGK1 were individually annealed and sub-cloned into the GV369 vector . The construct and the lentivirus packaging constructs (Genechem) were co-transfected to HEK-293T cells, generating PGK1-shRNA-expressing lentivirus. The latter was filtered, enriched and added directly to OB-6 osteoblastic cells or primary human osteoblasts . Following selection by puromycin  stable cells were established, with PGK1 silencing (over 90% knockdown efficiency) verified by qPCR and western blotting. Control cells were transduced with lentiviral scramble control shRNA (“sh-C”). For Nrf2 or Keap1 silencing, the Nrf2 shRNA or the Keap1 shRNA lentiviral particles  were individually added to cultured OB-6 cells. After puromycin selection the stable cells were established, with Nrf2 or Keap1 silencing verified by qPCR and western blotting assays.A set of two different shRNA oligonucleotides . The construct was tranduced to OB-6 osteoblastic cells (cultured at 60% cell confluence) by using the Lipofectamine 2000 protocol. The transfected cells were further subjected to fluorescence-activated cell sorting (FACS) GFP sorting, with single cells distributed to the 24-well plates for 2 more weeks. Stable cells were subjected to PGK1 expression screen. PGK1 KO was verified by qPCR and western blotting assays. Control cells were transduced with the CRISPR/Cas9 PX458-GFP construct with scramble non-sense sgRNA (“sg-C”).The targeted small guide RNA  was measured at the test-wavelength of 490 nm.At a density of 3 × 104 cells in each well). Following the applied DEX treatment a two-step simple LDH assay kit  was utilized to quantify LDH contents in the medium, always normalized to total LDH levels.OB-6 cells or primary osteoblasts were seeded into 12-well tissue-culture plates (at a density of 5 × 1026. OB-6 osteoblastic cells or primary human osteoblasts were seeded into 12-well tissue-culture plates (at a density of 5 × 104 cells in each well). Following the applied DEX treatment OB-6 cells or the primary osteoblasts were stained with JC-1  and tested immediately by a fluorescence spectrofluorometer at 550 nm. The representative JC-1 images, merging the green fluorescence image (at 550 nm) together with the red fluorescence image (at 650 nm), were presented as well.With mitochondrial depolarization JC-1 aggregating in mitochondria forms green monomers5 cells in each well). Following the indicated DEX treatment, a superoxide colorimetric assay kit  was applied to measure cellular superoxide contents. In brief, the superoxide detection reagent (100 µL/well) was added to cultured cells for 30 min, with the superoxide absorbance measured at 450 nm.OB-6 osteoblastic cells or primary human osteoblasts were seeded into six-well tissue-culture plates (at 1 × 105 cells in each well). We compared the ratio of reduced glutathione (GSH) with oxidized disulfide form glutathione (GSSG)27, using a previously-described protocol27.OB-6 cells or primary human osteoblasts were seeded into six-well tissue-culture plates (at 1 × 105 cells per well). Following the applied DEX treatment cell apoptosis was tested by Annexin V-PI assay, using the described protocol28.OB-6 cells or primary osteoblasts were seeded into six-well plates 29, with ELISA absorbance tested at 450 nm.OB-6 cells were seeded into 96-well plates at a density of 3 × 1021, from twelve (12) written-informed consent DEX-treated patients with femoral head resection, the necrotic femoral head tissues and surrounding normal femoral head tissues were collected, dissolved in the tissue lysis buffer, and tested by qPCR and western blotting. The clinical investigations were conducted according to the principles of Declaration of Helsinki. The protocols of this study were approved by Ethics Committee of Peking Union Medical College Hospital.As described in refs. p values < 0.05 were considered statistically significant28.The investigator was blinded to the group allocation during the experiments. Data were presented as means ± standard deviation (SD). Statistical analyses were performed in data with normal distribution by one-way analysis of variance (ANOVA) followed by multiple comparisons with Bonferroni’s post hoc test . The two-tailed unpaired T test (Excel 2007) was utilized when comparing two specific treatment groups. 31 were transduced with the lentiviral PGK1 shRNA , and selected by puromycin to establish the stable cell lines. Analyzing mRNA expression, by qPCR, show that PGK1 mRNA levels decreased over 95% in sh-PGK1-expressing stable cells (p < 0.05 vs. cells with non-sense control shRNA/“sh-C”)  Fig. . PGK1 prC”) Fig. . To examlls Fig. , whereaslls Fig. , indicatSignificantly, PGK1 silencing by sh-PGK1-S1 failed to further increase Nrf2 protein expression in OB-6 cells with pre-treatment of MG-132, a well-known proteasome inhibitor Fig. . In sh-PPGK1 mRNA and protein expression  potently downregulated ion Fig. . Furtherion Fig. , mRNA an15. On the contrary, activation of Nrf2 cascade can protect osteoblasts from DEX-induced oxidative stress15. We have shown that PGK1 silencing by targeted shRNA-induced Nrf2 cascade activation, we next tested whether it could protect osteoblasts from DEX.Previous studies have shown that DEX induces ROS production and significant oxidative injury in human osteoblasts, leading to following cell death and apoptosis33, in “sh-C” control OB-6 cells DEX treatment induced significant viability (MTT OD) reduction  treatment in control OB-6 cells induced significant mitochondrial depolarization   Fig. . As compLC) Fig. . FunctioLC) Fig. . FurtherLC) Fig. , cell deLC) Fig.  and apopLC) Fig.  were larIn the primary human osteoblasts, the applied CRISPR/Cas9-PGK1 KO construct led to PGK1 depletion Fig. , Nrf2 prHO1, NQO1, and GCLC was almost completely blocked by Nrf2 shRNA in OB-6 cells. Furthermore, the Nrf2 protein stabilization in ko-PGK1 cells was reversed by the Nrf2 shRNA , see Fig. RNA Fig. . The incRNA Fig. . In OB-6RNA Fig. , cell deRNA Fig.  and apopRNA Fig. . Such acRNA Fig. . These r19, we therefore hypothesized that Keap1 silencing should mimic PGK1 depletion-induced actions. OB-6 osteoblastic cells were transduced with the the lentiviral Keap1 shRNA, resulting in Keap1 depletion , when compared with its levels in the surrounding normal bone tissues (“S”). Importantly, PGK1 mRNA downregulated correlated with HO1 mRNA depletion in the necrotic femoral head tissues   Fig. . Togethe37. Keap1’s covalent modification will induce Keap1-Nrf2 disassociation and Nrf2 accumulation due to decreased Nrf2 ubiquitination37. After nuclear translocation, the stabilized Nrf2 initiates the transcription and expression of antioxidant and other cytoprotective genes through binding to ARE loci37. Recent studies have discovered a direct link between glycolysis and the Keap1-Nrf2 cascade activation38. PGK1 is an essential enzyme required for the ATP-generating step in the glycolytic pathway18. PGK1 inhibition or depletion caused methylglyoxal accumulation, thereby modifying Keap1 to form a methylimidazole crosslink19. This will lead to Keap1 dimerization, Nrf2 accumulation and Nrf2 signaling activation19.Keap1 has reactive cysteine residues functioning as the electrophile sensor of reactive speciesHO1, NQO1, and GCLC. Furthermore, CRISPR/Cas9-induced PGK1 KO also led to significant Nrf2 cascade activation in OB-6 cells and primary osteoblasts. These results indicate that PGK1 depletion could be a novel and efficient strategy to activate Nrf2 signaling in human osteoblasts.Here we demonstrated that PGK1 is functional expressed in OB-6 osteoblastic cells and primary human osteoblasts. PGK1 silencing, by targeted shRNA, resulted in Nrf2 cascade activation, leading to Nrf2 accumulation, nuclear translocation and expression of Nrf2-ARE-dependent genes, including 39. Studies have shown that DEX will induce free radical toxicity and oxidative injury to osteoblastic cells/osteoblasts, causing significant cell apoptosis and necrosis40. Conversely, ROS scavenging will efficiently protect osteoblastic cells/osteoblasts from DEX41. Here we show that PGK1 shRNA in OB-6 cells and primary osteoblasts potently inhibited DEX-induced ROS production, cell apoptosis and programmed necrosis. Similarly, PGK1 KO also exerted osteoblast cytoprotection against DEX-induced oxidative injury. Thus, targeting PGK1 can protect human osteoblasts from DEX through inhibiting oxidative stress (see the proposed signaling pathway in Fig. DEX usage is associated with increased risk of osteoporosis or even osteonecrosis in more than half of patients on long-term corticosteroid therapiesSignificantly, we show that Keap1-Nrf2 signaling activation is required for osteoblast cytoprotection by PGK1 depletion. In OB-6 cells PGK1 KO-induced cytoprotection against DEX was almost completely reversed by Nrf2 shRNA. In addition, Keap1 silencing, by targeted shRNA, activated Nrf2 signaling and protected OB-6 cells from DEX, mimicking PGK1 depletion-induced actions. More importantly, Keap1 shRNA-induced Nrf2 activation and anti-DEX osteoblast cytoprotection were not further augmented with PGK1 silencing. These results clearly show that activation of Nrf2 cascade by PGK1 depletion protected osteoblastic cells/osteoblasts from DEX-induced oxidative injury. Although the detailed mechanisms may warrant further characterizations.PGK1 mRNA and protein expression is significantly downregulated in the necrotic femoral head tissues of DEX-taking patients. This could be one reason of Nrf2 signaling inhibition in necrotic femoral head tissues, as reported by other studies32. We further show that downregulation of PGK1 correlated with depletion of HO1, a key Nrf2 pathway gene42, in necrotic femoral head tissues of DEX-taking patients. The underlying mechanism of PGK1 downregulation shall need more studies.One important finding of this study is that In summary we show that PGK1 depletion protects human osteoblasts from DEX via activation of Keap1-Nrf2 signaling cascade. Targeting PGK1-Nrf2 cascade could be a novel strategy to offer osteoblast cytoprotection against DEX-induced oxidative injury.


Recently, we have presented a scheme, termed “NKL-code”, which describes physiological expression patterns of NKL homeobox genes in early hematopoiesis and in lymphopoiesis including main stages of T-, B- and NK-cell development. Aberrant activity of these genes underlies the generation of hematological malignancies notably T-cell leukemia. Here, we searched for deregulated NKL homeobox genes in main entities of T-cell lymphomas comprising angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), adult T-cell leukemia/lymphoma (ATLL), hepatosplenic T-cell lymphoma (HSTL), NK/T-cell lymphoma (NKTL) and peripheral T-cell lymphoma (PTCL). Our data revealed altogether 19 aberrantly overexpressed genes in these types, demonstrating deregulated NKL homeobox genes involvement in T-cell lymphomas as well. For detailed analysis we focused on NKL homeobox gene MSX1 which is normally expressed in NK-cells. MSX1 was overexpressed in subsets of HSTL patients and HSTL-derived sister cell lines DERL-2 and DERL-7 which served as models to characterize mechanisms of deregulation. We performed karyotyping, genomic and expression profiling, and whole genome sequencing to reveal mutated and deregulated gene candidates, including the fusion gene CD53-PDGFRB. Subsequent knockdown experiments allowed the reconstruction of an aberrant network involved in MSX1 deregulation, including chromatin factors AUTS2 and mutated histone HIST1H3B(K27M). The gene encoding AUTS2 is located at chromosome 7q11 and may represent a basic target of the HSTL hallmark aberration i(7q). Taken together, our findings highlight an oncogenic role for deregulated NKL homeobox genes in T-cell lymphoma and identify MSX1 as a novel player in HSTL, implicated in aberrant NK- and T-cell differentiation. All types of lymphocytes are generated in the course of hematopoiesis which starts with hematopoietic stem cells (HSC) and the derived common lymphoid progenitors (CLP) in the bone marrow. The subsequent differentiation of B-cells and NK-cells persists in this compartment while early T-cell progenitors (ETP) move into the thymus to complete their differentiation. The developmental steps in early hematopoiesis and in lymphopoiesis are primarily regulated at the transcriptional level . AccordiHomeobox genes encode TFs which are involved in basic operations of cell, tissue, and organ differentiation in the embryo and in the adult. NKL homeobox genes represent a subclass of this group and comprise 48 members, including NKX2-3 and NKX2-5 which operate as master genes in the developing spleen and heart, respectively . RecentlMechanisms of NKL homeobox gene deregulation include recurrent chromosomal rearrangements juxtaposing NKL genes with those encoding the T-cell receptors (TCR) or BCL11B, aberrant activities of signalling pathways including the BMP-pathway, failed downregulation of upstream factors normally operating in early stages of hematopoiesis only, and aberrant alterations of chromatin components including histones and AUTS2 . ChromatHere, we extended our analysis of aberrantly activated NKL homeobox genes to mature T-cell and NK-cell neoplasms. According to the WHO classification, this group of malignancies comprises several entities including the most common type peripheral T-cell lymphoma (PTCL) and the rare type hepatosplenic T-cell lymphoma (HSTL) , 21. ManN = 11) while ATLL and HSTL each showed the lowest number of deregulated genes (N = 6). Collectively, our data demonstrate that NKL homeobox gene deregulation is a frequent event in both, T-cell leukemia and T-cell lymphoma.NKL homeobox genes are silent in normal mature T-cells and play a basic oncogenic role in T-cell leukemia if aberrantly activated , 22. To In the following, we focused our study on MSX1. This NKL homeobox gene is physiologically expressed in early immature lymphocytes including CLPs and B-cell progenitors in addition to mature NK-cells but downregulated in terminal development of B- and T-cells , 8, 19. Subsequent RQ-PCR analysis performed in selected T-cell lymphoma cell lines in comparison to samples of primary NK-cells, T-cells and HSCs confirmed aberrant overexpression of MSX1 in DERL-2 and demonstrated even higher levels in sister cell line DERL-7 . In DERLCluster analysis using gene expression profiling data of cell lines derived from T-cell lymphomas, T-ALL, and NK-cell leukemia revealed the greater proximity of DERL-2/7 to NK-cells than to T-cells . This fiETS1, ID2 and TBX21/T-bet represent master regulators of NK-cell development . To analIn T-ALL aberrant expression of NKL homeobox genes is frequently associated with their chromosomal/genomic rearrangement . To examGenomic profiling identified several copy-number alterations in DERL-2/7. However, this analysis confirmed wild type configurations of the MSX1 locus in both cell lines . In contWhole genome sequencing of DERL-2 and DERL-7 allowed the identification of both, chromosomal rearrangements and gene mutations. However, while these data confirmed the absence of rearrangements at MSX1 they revealed mutations in the genes HIST1H3BK27M), KDM7A(P897L), SETD2P1962L), STAT5B(N642H) and TLE1(P287A) in both cell lines. These mutations were confirmed by Sanger-sequencing  and match exome-sequencing data obtained from DERL-2/7 and HSTL patients [M, KDM7A, exclusive to DERL-2. Genome sequencing showed that this translocation resulted in the fusion of CD53 at 1p13 to PDGFRB at 5q32 . The breakpoints were located in intron 2 of CD53 and intron 11 of PDGFRB. Expression of the resultant fusion gene CD53-PDGFRB was confirmed by RT-PCR . RQ-PCR The fusion-transcript consists of two CD53 exons at the 5′-end and of twelve PDGFRB exons at the 3′-end which lacks the encoded ligand-binding domain but retains the tyrosine-kinase domain. This aberrant configuration of PDGFRB has been detected in combination with other fusion partners in myeloid malignancies supporting an oncogenic function for this receptor gene . Of noteTo investigate whether PDGFRB is involved in MSX1 regulation in HSTL we performed siRNA-mediated knockdown experiments targeting sequences located in exon 17 which are present in both wild type PDGFRB and the fusion-transcript. Interestingly, reduction of PDGFRB resulted in elevated MSX1 transcript levels in DERL-2 while this procedure reduced MSX1 expression in DERL-7 . TreatmeAberrant expression levels of histones and chromatin-modifiers have been reported to deregulate the activity of NKL homeobox genes in T-ALL and B-cell lymphomas, highlighting the significance of this deregulatory mechanism , 15. Thehttps://www.ncbi.nlm.nih.gov). This comparison revealed a significant correlation of PDGFRA with MSX1 expression . Finally, we used dataset GSE19067 to compare four HSTL patients (MSX1-high) with seven NKTL patients (MSX1-low). This analysis revealed elevated expression levels of NKX2-3, TLX1, BAMBI and ENG in MSX1-high HSTL patients . Taken together, these data indicated four categories of gene candidates probably involved in MSX1 (de)regulation: (1) PDGF-signalling pathway , (2) BMP-signalling pathway , (3) homeobox genes , and (4) TLE corepressors (TLE1). Interestingly, most of these candidate genes have been previously reported to deregulate MSX1 expression in lymphoid malignancies [To identify additional MSX1-regulators in HSTL we performed comparative expression profiling analyses. First, we compared DERL-2 (MSX1 low) and DERL-7 (MSX1 high) to reveal specific differences among the top-1000 differentially expressed genes: DERL-2 expressed enhanced levels of BMP4, CD8A and SLFN13 while DERL-7 expressed CD3D, CD3E, PDGFD and TLE1 at higher levels (Supplementary Table 1). Next, we compared DERL-7 (MSX1 high) and three NK-cell lines , demonstrating in DERL-7 higher levels of NKX2-2, OTX2, PDGFD, PITX1, SIX1 and TLE1 and lower levels of CDKN2A, SLFN13 and STAT5B (Supplementary Table 2). For comparison of HSTL patients we analyzed dataset GSE57944 (including two MSX1-high and two MSX1-low samples) and used an associated R-based online-tool at GEO , which resulted in formation of the fusion gene CD53-PDGFRB. Several PDGFRB fusion genes have been detected in myeloid and rarely in lymphoid maligancies , 40, 41.Post-translational modifications of histones are basic to gene regulation. In particular, tri-methylation of histone H3 at position K27 is performed by EZH2 and effects transcriptional repression. H3K27-mutations including H3(K27M) inhibit EZH2 activity and thereby decrease global levels of repressive H3K27me3 , 45. ThuOur results revealed the roles of homeobox genes NKX2-2 and PITX1 in HSTL which serve as oncogenes in T-ALL , 31, 54.In this study, we also identified several additional cancer genes: STAT5B mutated at N642H which displays oncogenic aberrations in both T-cell lymphoma and T-ALL , 56, 57;HSTL-derived cell lines DERL-2/7 are malignant T-cells and express apposite markers . FurtherTumors are not constant. They evolve by the generation of genetic diversity and subsequent natural selection of subclones best able to resist host defence and treatment . TherefoThe phenomenon of tumor evolution has been described in other cell lines as well including Hodgkin lymphoma sister cell lines HDLM-1 and HDLM-2, diffuse large B-cell lymphoma subclones U-2932-R1 and U-2932-R2, and cutaneous T-cell lymphoma sister cell lines MAC-1, MAC-2A, MAC-2B –67. ThesTaken together, our data revealed deregulated NKL homeobox gene MSX1 in HSTL. By analyzing aberrant mechanisms involved in MSX1 expression we recognized differences in the sister cell lines DERL-2 and DERL-7 which might represent consequences of clonal selection or intratumoral diversity. In the evolutionary course of this tumor the malignant cells shifted their identity from the T-cell status back to progenitor cells, gaining NK-cell characteristics. These results may support the understanding of the biology of HSTL tumors and may assist to develop novel therapeutic options in the future.https://www.ncbi.nlm.nih.gov). We exploited datasets GSE6338, GSE19069, GSE57944 and GSE19067 for examinations of T-cell lymphoma entities. In datasets GSE6338 and GSE19069 outliers and/or statistically significantly higher values of the median was defined as overexpression. In datasets GSE57944 and GSE19067 we set a cut-off at 3.0 and 8.0, respectively, to identify patient samples showing overexpression.Public expression profiling datasets used in this study were generated by U133 Plus 2.0 gene chips from Affymetrix and obtained from Gene Expression Omnibus  using limma and affy packages. The generation of the dendrogram was performed via hierarchical clustering by the Ward‘s method which was applied on the Euclidean distance matrix.For clustering of cell lines according to gene expression profiling data we used datasets generated by Prof. Andreas Rosenwald , by Dr. Robert Geffers , and retrieved from GEO datasets GSE128302 (T-cell lymphomas), GSE87334 (T-ALLs), GSE19067  and GSE53478 (NK-92). After RMA-background correction and quantile normalization of the spot intensities, gene expression values were subsequently log2 transformed. Data processing was performed via R/Bioconductor .For creation of heat maps showing selected values of gene expression profiling data we used the software CLUSTER (version 2.11) and TREEVIEW (version 1.60) originally developed by Michael Eisen  at 350 V for 10 ms. Electroporated cells were harvested after 20 h cultivation. Cells were variously treated for 16 h with 20 ng/ml recombinant BMP4, with 1 or 10 ng/ml recombinant PDGFD , with 100 μM Dasatinib , with 5 μM BMP receptor inhibitor dorsomorphin (DM)  dissolved in dimethyl sulfoxide (DMSO), with 10 μM EZH2-inhibitor DZNep , or with 10 μg/ml histone deacetylase inhibitor trichostatin A (TSA) (Sigma).The cell lines DERL-2, DERL-7, KHYG-1, NK-92, YT, and LOUCY were obtained from the DSMZ  and cultivated as described elsewhere , 68. TheT-Test and the calculated p-values were indicated by asterisks .Total RNA was extracted from cultivated cell lines using TRIzol reagent . Primary human total RNA was commercially obtained. We used RNA from T-cells and NK-cells obtained from Biochain/BioCat , and RNA from HSCs obtained from Miltenyi Biotec . cDNA was synthesized using 5 μg RNA, random priming and Superscript II (Invitrogen). Real-time quantitative (RQ)-PCR analysis was performed using the 7500 Real-time System and commercial buffer and primer sets . Quantification of MSX1 was performed as described recently . For norReverse-transcription (RT)-PCR analysis was performed using Taq-DNA polymerase (Qiagen) and thermocycler TGradient . The oligonucleotides were obtained from Eurofins MWG  and their sequences were as follows: CD53-for 5′-TCTGTGTTACCAGCCTTGTCTCG-3′, CD53-rev 5′-GACAAACACATTGCCCAGCGTG-3′, PDGFRB-for 5′-ACACTGCGTCTGCAGCACGTGG-3′, PDGFRB-rev 5′-GGAGTCATAGGGCAGCTGCATG-3′. The generated PCR products were analyzed by agarose gel electrophoresis using Gene Ruler 100 bp Plus (Thermo Fisher) as marker.6 cells which were washed in PBS and subsequently incubated in 1 ml medium for 24 h.Western blots were generated by the semi-dry method. Protein lysates from cell lines were prepared using SIGMAFast protease inhibitor cocktail (Sigma). Proteins were transferred onto nitrocellulose membranes  and blocked with 5% dry milk powder dissolved in phosphate-buffered-saline buffer (PBS). The following antibodies were used: MSX1 (R & D Systems), alpha-Tubulin (Sigma), PDGFRB (R & D Systems), phospho-PDGFRB , NKX2-2 (Aviva Systems Biology) and PITX1 . For loading control blots were reversibly stained with Poinceau (Sigma) and detection of alpha-Tubulin (TUBA) was performed thereafter. Secondary antibodies were linked to peroxidase for detection by Western-Lightning-ECL . Documentation was performed using the digital system ChemoStar Imager . PDGFD and BMP4 were quantified in the medium by ELISA using according Quantikine ELISA kits from R & D Systems. Samples were obtained by harvesting supernatants of 1x10The karyotypes of DERL-2 and DERL-7 were generated as described previously . For genhttps://www.bioinformatics.babraham.ac.uk/projects/fastqc) and trimmed via fastq-mcf (ea-utils 1.04.807). The data have been deposited in the ArrayExpress database at EMBL-EBI (https://www.ebi.ac.uk/arrayexpress) via accession number E-MTAB-7734. For detection of gene mutations the reads were aligned by STAR (version 2.5.3a) to the Gencode Homo sapiens genome (version 26) and converted/sorted via samtools (version 0.1.19) [https://www.bioconductor.org). Genomic structural variants were detected via seeksv (version 2.0) and lumpy [Genomic sequencing was performed as follows: Standard genomic library preparation and sequencing were conducted at GATC Biotech . The libraries were sequenced on Illumina HiSeq2500  with >300 million reads per sample for a coverage of 30-fold. Reads were quality controlled via FastQC  . Duplica 0.1.19) , 76. Mutnd lumpy , 79.For confirmation of identified mutations we performed Sanger sequencing of cDNA samples. DNA-fragments were generated by PCR using the following oligonucleotides: HIST1H3B-for 5′-ATGGCTCGTACTAAACAGACAGC-3′, HIST1H3B-rev 5′-AGAGCCTTTGGGTTTTAAGACTG-3′, KDM7A-for 5′-GTAGGAATTATGTGGACAGCAG-3′, KDM7A-rev 5′-TATACACACAAACTGCTCCAGG-3′, SETD2-for 5′-CATGGACAGTGCAATCTCTGATG-3′, SETD2-rev 5′-AACTGTCCAGGAGTTTGGTGGC-3′, STAT5B-for 5′-AGGACGGAATTACACTTTCTGG-3′, STAT5B-rev 5′-ATCTGTGGCTTCACGTATCCATC-3′, TLE1-for 5′-GTGATGGTGACAAAAGCGATGAC-3′, TLE1-rev 5′-CAAAAGGAGCAGGATATGGGCC-3′. PCR products were treated using exonuclease 1 and alkaline phosphatase Illustra ExoProStar according to the recommended protocol for a 5 μl aliquot . The sequencing reactions were performed using BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher) for 25 cycles in a Veriti Thermal Cycler (Thermo Fisher). For purification we used CleanSEQ reagent in combination with the Agencourt CleanSEQ magnetic plate . The beads were eluted with 40 μl HiDi formamide and applied to the ABI 24 capillary 3500XL automated DNA sequencer (Thermo Fisher).HindIII/BamHI and EcoRI sites, respectively, of the expression vector pcDN A3 downstream of the CMV enhancer. The oligonucleotides used for the amplification of the NKX2-2 site were obtained from Eurofins MWG . Their sequences were as follows: MSX1-for 5′-CC AAGCTTCAGGCAGATCTTGCATCTCC-3′, MSX1-rev 5′-ATGGATCCTTATCCTAGGAGAAAGACATACTAT TAAC-3′, HOXA9-for 5′-TGGCATTAAACCTGAA CCGC-3′, HOXA9-rev 5′-ACTCTTTCTCCAGTTC CAGG-3′. Introduced restriction sites used for cloning are underlined. Transfections of gene expression construct for NKX2-2  into NIH-3T3 cells was performed using SuperFect Transfection Reagent (Qiagen). Commercial HOXA9 and TBP assays (Thermo Fisher) were used for RQ-PCR to quantify the spliced reporter-transcript, corresponding to the regulator activity.For creation of a reporter gene construct we combined a reporter with a regulatory genomic fragment derived from the upstream region of MSX1, containing a consensus binding site for NKX2-2 . We clon


Scientific Reports 10.1038/s41598-018-29773-0, published online 27 July 2018Correction to: This Article contains an error in Figure 3E, where the HFD+SRT1720 label is missing. The correct Figure 3 appears below as Figure


Scientific Reports 10.1038/s41598-018-37514-6, published online 04 February 2019Correction to: The Acknowledgements section in this Article was omitted. The Acknowledgements section should read:“This work was supported by Zhejiang Provincial Natural Science Foundation (Grant Number LGF18H180011) and Jinhua Science and Technology Bureau (Grant Number 2017-3-004)”.


Drosophila imaginal discs. We find that reducing the levels of the ferritin heavy chain in the larval wing discs leads to drastic growth defects, whereas light chain depletion causes only minor defects. Mutant cell clones for the heavy chain lack the ability to compete against Minute mutant cells. Reactive oxygen species (ROS) accumulate in wing discs with reduced heavy chain levels, causing severe mitochondrial defects and ferroptosis. Preventing ROS accumulation alleviates some of the growth defects. We propose that the increased expression of ferritin in hippo mutant cells may protect against ROS accumulation.The interplay between signalling pathways and metabolism is crucial for tissue growth. Yet, it remains poorly understood. Here, we studied the consequences of modulating iron metabolism on the growth of  Despite being vital, the role of metals in biology is often overlooked. Specifically, how iron storage contributes to development remains unclear. Here, we dissected the function of the cellular iron storage complex, Ferritin, during development of a model organ, the fly wing. We took a genetic approach to uncover the role of both the heavy and light chains of Ferritin. Targeting the heavy chain consistently produced drastic growth defects in larval discs and adult wings. Moreover, lower levels of the heavy chain led to formation of reactive oxygen species (ROS) and preventing ROS accumulation alleviated the accompanying growth defects. We also observed hallmarks of ferroptosis, an iron dependent non-apoptotic cell death, upon knockdown of the heavy chain. By contrast, reducing the levels of the light chain was tolerable. This is surprising because the individual components of Ferritin were thought to function exclusively as part of the complex. Yet, the heavy chain alone encompasses the ferroxidase center, which is essential for iron loading. All together, we propose that the Ferritin heavy chain functions as an antioxidant and protects the developing organs from ferroptosis. The latter reaction generates reactive oxygen species, which damage biological macromolecules. Iron-binding proteins, such as ferritin, ensure that iron levels in a cell are tightly controlled. The insect ferritin complex is formed by 24 subunits of the heavy and the light chain proteins in a 1:1 ratio; it is the main iron storage facility of the cell  in cacodylate buffer followed by 1 hour in 1% (wt/vol) tannic acid in 100 mM cacodylate buffer, then 30 minutes in 2% (wt/vol) Osmium tetroxide followed by 1% (wt/vol) uranyl acetate for 2h at room temp. After the dehydration cycles, and incubations in Epon-Araldite mix, samples were flat embedded and cured for 24h at 60°C [Wing discs were dissected in PBS and immediately fixed with 1% paraformaldehyde, 2.5% glutaraldehyde in 0.1M cacodylate buffer for two hours at room temperature. The samples were incubated for 1 hour in 2% (wt/vol) osmium tetroxide and 1.5% (wt/vol) K at 60°C , 44.Polymerized flat blocks were trimmed using 90° diamond trim tool  and using 35° diamond knife  mounted on Leica UC6 microtome , 70 nm sections were collected on formvar-coated slot grids . For sectioning, samples were carefully oriented to obtain the relevant parts of the wing blade, indispensable for the reliable data interpretation , 45. TEMS1 Fig(a) Row normalized heat-maps of top 15 and bottom 5 of the 120 genes that are induced in warts (wts) mutant discs with a cut-off of 2,3-fold induction. The color key is coded in log2. Green bars represent average expression levels. (b-c) RT-qPCR analysis of (b) Fer1HCH and (c) Fer2LCH mRNA levels in wing discs that are nearly fully mutant for a null wts allele at day 5 and day 9. In such discs, wts mutant cells take over the disc and cause tissue overgrowth. The genotypes are:control: y ubx-flp / y w; FRT82B M(3) ubiGFP / FRT82B at day 5wts: y ubx-flp / y w; FRT82B M(3) ubiGFP / FRT82B wts149 at day 5 and day 9.(TIF)Click here for additional data file.S2 Fig(a)Nub-Gal4 driven knockdown of Fer1HCH and Fer2LCH using all available UAS-RNAi lines from the Vienna Drosophila Resource Center (VDRC). Note that the two strong RNAi lines (102406 and 12925) cause lethality at pupal stage and the few escapers have no wings. Representative adult wings and quantifications of wing areas for the indicated genotypes are shown. (b) Knockdown of Fer1HCH and Fer2LCH modify the overgrowth phenotype of Nub-Gal4 > UAS-hippo-RNAi wings to differing extents. Statistical significance is indicated as ns: p>0.05, *: p≤0.05, **: p≤0.01, ***: p≤0.001, ****: p≤ 0.0001.(TIF)Click here for additional data file.S3 Fig(a-d’) EdU (purple in a-d and gray in a’-d’) profiles upon knockdown of Fer1HCH with (b) a weak (VDRC 49537) and (c) a stronger (VDRC 102406) RNAi line in the posterior compartments (right hand side). (d) Fer2LCH knockdown in the posterior compartment does not influence the EdU pattern. Ptc antibody staining in green marks the A/P boundary. (e-h’) Activated caspase expression revealed by anti-Dcp-1 (purple in e-h and gray in e’-h’) in discs with hh-gal4 (posterior specific) driven knockdown of Fer1HCH with (f) a weak (VDRC 49537) and (g) a stronger (VDRC 102406) RNAi line. (h) Fer2LCH knockdown in the posterior compartment does not induce caspase activation. Ptc antibody staining in green marks the A/P boundary. (i)TUNEL staining in day 5 discs mosaic for Fer1HCH451 (two discs are shown), Fer2LCH35, and the Df(3R)Fer deletion. GFP (green) marks the Minute cells and Fer mutant cells are unmarked. TUNEL is shown separately in gray below each panel. Apoptosis is induced in discs mosaic for the heavy chain in a non-cell-autonomous and variable fashion. The genotypes are:wt: y ubx-flp / y w; FRT82B M(3) ubiGFP / FRT82Bfer1: y ubx-flp / y w; FRT82B M(3) ubiGFP / FRT82B Fer1HCH451fer2: y ubx-flp / y w; FRT82B M(3) ubiGFP / FRT82B Fer2LCH35Df(3R): y ubx-flp / y w; FRT82B M(3) ubiGFP / FRT82B Df(3R)Fer.(TIF)Click here for additional data file.S4 FigRepresentative eye discs of indicated genotypes at day 5. All discs are shown at the same scale. Scale bar in (h) is 50 microns. (i and j) show quantifications of area occupied by the mutant cells as a percentage of the whole disc area. Genotypes are:y eyflp; FRT82B ubiGFP / FRT82Ba) y eyflp; FRT82B ubiGFP / FRT82B Fer1HCH451b) y eyflp; FRT82B ubiGFP / FRT82B Fer2LCH35c) y eyflp; FRT82B ubiGFP / FRT82B Df(3R)Ferd) y eyflp; FRT82B M(3) ubiGFP / FRT82Be) y eyflp; FRT82B M(3) ubiGFP / FRT82B Fer1HCH451f) y eyflp; FRT82B M(3) ubiGFP / FRT82B Fer2LCH35g) y eyflp; FRT82B M(3) ubiGFP / FRT82B Df(3R)Fer.h) (TIF)Click here for additional data file.S5 Fig(a-e) Representative third instar wing discs with flp-out clones expressing (a) UAS-GFP, (b) UAS-GFP + UAS-Fer1HCH-RNAi-s (VDRC102406), (c) UAS-GFP + UAS-Fer1HCH-RNAi-s + UAS-DIAP1, (d) UAS-GFP + UAS-Fer1HCH- RNAi-s + UAS-p35, and (e) UAS-GFP + UAS-Fer1HCH- RNAi-s + UAS-Yki. GFP (green) marks the modified clonal patches and DAPI (blue) labels nuclei. All samples were treated in parallel and imaged at the same settings. (f) Shows quantifications of (left) clone occupancy , and (right) overall disc area normalized to the average control disc size. Five discs per genotype were measured; error bars represent standard deviation. The rescue by UAS-Yki (e vs b) is significant (p≤0.001). The rescue by UAS-DIAP1 and p35  are merely significant (p≤0.05).(TIF)Click here for additional data file.S6 Fig(a-c) Apical to basal cross-section of tiled TEM images of representative discs with ptc-Gal4 driven expression of indicated UAS-RNAi lines. (a) Discs expressing UAS-Fer2LCH have an overall wild-type appearance. (b) Discs with Fer1HCH (VDRC 102406) knockdown are thinner and disorganized. (c) Discs with ectopic Fer1HCH look nearly normal. All TEM composites (a-c) are shown at the same magnification, scale bar in (b) is 2nm. (d) Representative third instar discs of indicated genotypes used for TEM analysis, shown at the same magnification. Scale bar is 50uM. (e-f) Shows examples of mitochondria that were classified as normal (e) or abnormal (f) for quantification shown in (TIF)Click here for additional data file.


Caenorhabditis elegans vulval cell fate patterning – an environmentally sensitive process, with a nevertheless robust output. Past research has shown many vulval mutants affecting EGF-Ras-MAPK, Delta-Notch and Wnt pathways to be suppressed by environmental factors, such as starvation. Here we aimed to resolve previous, seemingly contradictory, observations on how starvation modulates levels of vulval induction. Using the strong starvation suppression of the Vulvaless phenotype of lin-3/egf reduction-of-function mutations as an experimental paradigm, we first tested for a possible involvement of the sensory system in relaying starvation signals to affect vulval induction: mutation of various sensory inputs, DAF-2/Insulin or DAF-7/TGF-β signaling did not abolish lin-3(rf) starvation suppression. In contrast, nutrient deprivation induced by mutation of the intestinal peptide transporter gene pept-1 or the TOR pathway component rsks-1  very strongly suppressed lin-3(rf) mutant phenotypes. Therefore, physiologically starved animals induced by these mutations tightly recapitulated the effects of external starvation on vulval induction. While both starvation and pept-1 RNAi were sufficient to increase Ras and Notch pathway activities in vulval cells, the highly penetrant Vulvaless phenotype of a tissue-specific null allele of lin-3 was not suppressed by either condition. This and additional results indicate that partial lin-3 expression is required for starvation to affect vulval induction. These results suggest a cross-talk between nutrient deprivation, TOR-S6K and EGF-Ras-MAPK signaling during C. elegans vulval induction.Studying how molecular pathways respond to ecologically relevant environmental variation is fundamental to understand organismal development and its evolution. Here we characterize how starvation modulates  Drosophila and C. elegans  although activities and interactions of underlying molecular signaling pathways are environmentally sensitive   vulval cell fate by activating the EGF-Ras-MAPK pathway in P6.p, which receives the highest dose of this signal     and 1B. osterior  and 1B. C. elegans vulval cell fate patterning to environmental perturbations, multiple reports demonstrate that this process is nevertheless responsive to environmental and physiological inputs  were found to be suppressed by starvation and/or dauer passage . This negative starvation effect requires the G-protein-coupled receptor SRA-13 to modulate EGF-Ras-MAPK activity  and let-23/egfr(rf) mutations. Genetic analyses reported in this study suggest that this starvation signal acts at the level or upstream of LET-23/EGFR  animals.-23/EGFR . The posand P7.p . In addiIn summary, starvation experienced during larval growth may have both positive  and negaC. elegans vulval cell fate patterning. In particular, we ask how sensory perception of external starvation conditions vs. the  animal’s nutrient status affect vulval induction.Addressing these open questions, we have aimed here to quantify and characterize more precisely how starvation affects E. coliOP50  carrying a lawn of oliOP50 . Animalsegl-30(ad805),che-3(e1124)LGI: let-23(sy1),sra-13(zh13)LGII: daf-7(e1372),mpk-1(ku1),daf-2(e1370),lin-39(n2110),rsks-1(ok1255),zhIs4[lip-1::GFP]LGIII: lin-3(e1417),lin-3(n378),lin-3(mf75)LGIV: arIs92LGV: daf-12(rh61rh411),bar-1(mu63),bar-1(ga80),sem-5(n2019),pept-1(Ig601),osm-5(p813)LGX: lin-3(mf75) allele is a recently described tissue-specific transcriptionally null allele  at the beginning of experiments. Individuals examined in different environments or of different strains were always scored in parallel and derived from populations kept in identical environmental conditions over at least two generations.i.e., unseeded NGM plates containing 1mg/ml of ampicillin to prevent bacterial growth. After 48 hr, starved animals were transferred back to regular NGM plates seeded with E. coliOP50 and the vulval phenotype was scored when animals had reached the early or mid L4 stage (approximately 15-20 hr later). Control animals were kept on NGM plates seeded with E. coliOP50 from L1 to L4. This starvation treatment drastically reduced, yet did not completely stop, developmental progression of worms: after 48 hr most animals had developed into the early to mid-L3 individuals. Note that this starvation treatment did not induce dauer formation.For starvation assays, L1 larvae were grown on standard NGM plates until they reached the mid L2 stage (23 hr after L1 transfer back on NGM seeded plates) unless mentioned otherwise. At this stage, animals were washed three times with sterile M9 buffer and transferred on starvation plates, HT115 bacterial strain carrying the empty RNAi expression vector L4440 served as a negative control. We used RNAi plates composed of standard NGM with 50μg/ml of ampicillin and 1mM of IPTG. Late L2/ early L3 individuals were transferred on RNAi bacteria. The vulval phenotype was scored in L4 individuals of the F1 generation. RNAi clones were selected from Ahringer’s RNAi library , let-363  and rsks-1 .RNAi by bacterial feeding was performed as described by pept-1 RNAi we used previously generated transgenic strains containing integrated transcriptional reporter constructs: the JU480 strain carries the egl-17::cfp-LacZ transgene (EGF-Ras-MAPK activity reporter) derived from the strain GS3582 (AH142 strain carries the lip-1::gfp transgene (Delta-Notch activity reporter) (pept-1 RNAi (K04E7.2 clone) or control (L4440 empty vector) plates. EGF-Ras-MAPK and Delta-Notch pathway activities were measured in lethargus L2/early L3 of the F1 generation.To quantify EGF-Ras-MAPK and Delta-Notch pathway activities in response to We quantified reporter gene activity in VPCs as previously described  mounted onto an upright Zeiss AxioImager M1 with a Lumen 200 metal arc lamp (Prior Scientific). Each Z-stack was composed of 30 images spanning 20.3 µm (0.7 µm in between two acquisitions) on each filter channel. Finally, spot quantification was performed using a custom-made Matlab routine optimized for the anchor cell. Only worms with gonad length >50 µm were included in the quantifications.L3 stage animals were processed to quantify e.g., Box-Cox- or log-transformed) when necessary to meet the assumptions of ANOVA procedures   procedure was used. Statistical tests were performed using the software programs JMP v13 or SPSS v19.Data were transformed  . For posAll strains generated in this study are available upon request. The authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article.e.g., animals were exposed to starvation conditions for different periods of time and at different developmental time points (lin-3(rf) mutants known to be affected by starvation conditions (lin-3(n378) animals (lin-3(n378) animals to different time periods of starvation (0-120h) at the mid L2 stage caused strong suppression of the Vulvaless phenotype when starved for 48 hr or more, low suppression after 19-24h of starvation, and no suppression after a brief (2h) exposure to starvation or early L1 starvation (12h) in liquid medium  mutants affects vulval cell lineages, we further scored cell fate patterns in mid-L4 individuals that had been exposed to 48 hr of starvation at the mid-L2 stage. The Vulvaless phenotypes of lin-3(n378) and lin-3(e1417) were strongly suppressed by starvation (lin-3(n378) compared to lin-3(e1417) , the proportion of individuals with complete induction increased from 12% in food conditions to 50% after starvation, and most of these individuals showed a correct 2°-1°-2° pattern for P5.p to P7.p  -2H. Thes to P7.p  and 2E. nduction .vs. 48-120 hr) had different effects, with prolonged starvation periods causing increased suppression (clk-1 (a gene necessary for the synthesis of ubiquinone) that causes a twofold increase in developmental time (lin-3(n378)  reduces vulval induction of lin-3(e1417) in food conditions (2I).We hypothesized that the prolonged developmental time in the starvation environment could affect the output of the signaling network. Applied starvation treatments caused a strong delay in development: animals remained in the L2 stage throughout the 48-hr starvation period . In addition, we observed that individuals starved for only short time period (2 hr) showed no suppression while longer starvation periods (19-24 hr pression . If the pression . However-3(n378) . Thus, alet-60/ras(gf) was significantly reduced after starvation of late L1 larvae, indicative of a negative starvation effect on vulval induction. Moreover, this study showed that mutation of the G-protein-coupled receptor sra-13 or mutations causing sensory signaling defects, such as osm-5(p813) and che-3(e1124), abolished starvation sensitivity of let-60(gf) animals, implying that observed starvation effects acted on vulval induction via components of the sensory system. We therefore tested whether disruption of the same sensory signaling components also inhibits starvation effects on vulval induction in lin-3(rf) mutants. However, we observed that starvation still significantly increased vulval induction of lin-3(n378) in sra-13(zh13), osm-5(p813) and che-3(e1124) backgrounds, comparable to the results of the lin-3(n378) single mutant (lin-3(n378) mutants.To understand how starvation stimuli are transduced to affect vulval induction, we first tested for an implication of the sensory system in relaying external starvation conditions. e mutant . Therefoe.g., during dauer formation (daf-7(e1372); lin-3(e1417); and starvation still significantly increased vulval induction in this double mutant (DAF-2/Insulin receptor caused by the daf-2(e1370) mutation did not mimic starvation effects on lin-3(n378) mutants in food conditions (daf-2(e1370) lin-3(n378) double mutants was significantly lower than in the single lin-3(n378) mutant, both in food and starvation conditions. This result is consistent with previous observations that reduced Insulin signaling may lower vulval induction in sensitized backgrounds, such as let-60/Ras(gf)   vulval induction  mutants, in both food and starvation conditions. Yet, in our experiments, starvation nevertheless increased vulval induction of daf-2(e1370); lin-3(n378) animals, suggesting that the negative starvation effect  pathway  (lin-3(n378) animals in food conditions  (lin-3(n378) even more strongly (lin-3(rf), consistent with the idea that TOR pathway components and targets mediate starvation effects on vulval induction. We further tested whether RNAi knock-down of an additional TOR pathway member daf-15 (RAPTOR) affects vulval induction using the lin-3(n378) mutant. RNAi of daf-15 did not cause any suppression of the Vulvaless phenotype of lin-3(n378)  , signifinditions . Moreovestrongly . These R-3(n378) .lin-3(n378) with rsks-1(ok1255) and pept-1(lg601), respectively. The two double mutants exhibited extremely strong suppression of the lin-3(n378) Vulvaless phenotype (lin-3(n378); pept-1(lg601) where mean vulval induction approached wild type levels (2.93 ± 0.03 cells induced) (lin-3(n378) individuals  background . Of note, however, neither pept-1(lg601) or rsks-1(ok1255) single mutants exhibited vulval hyperinduction or other vulval defects (N > 300/strain). As compromised PEPT-1 activity leads to strongly nutrient-deprived animals, even in a food-rich environment (lin-3(rf) animals  , and 70%ividuals  and 3C.  animals .pept-1(lg601) primarily acts through TOR signaling ; lin-3(n378) animals: pept-1 RNAi increased vulval induction in a daf-2(e1370); lin-3(n378) double mutants, but to a lesser extent than in lin-3(n378) single mutants (daf-2(e1370); lin-3(n378) animals  vulval induction compared to disruption of RSKS-1 activity ; lin-3(n378) double mutants exposed to either pept-1 RNAi or starvation exhibited a slight, but significant increase in vulval induction compared to control animals   ; Table 2and P7.p ; Table 2pathways . Observepept-1 RNAi on several components of the core EGF-Ras-MAPK cascade using various reduction-of-function mutants (lin-3(rf) mutations, pept-1 RNAi suppressed, to different degrees, the Vulvaless phenotypes of let-23(sy1), sem-5(n2019) and mpk-1(ku1) ; lin-3(n378) that exhibits a fully penetrant Vulvaless phenotype, indicating absence or very low levels of basal Ras activity (let-23(sy1); lin-3(n378) animals treated with pept-1 RNAi remained virtually unaltered compared to controls . Very similarly, a recently constructed tissue-specific null allele of lin-3/EGF, mf75 .To further characterize how physiological starvation interacts with vulval induction to modulate inductive levels, we characterized effects of  mutants . In addik-1(ku1) . Althougactivity . Vulval controls , with onpept-1 mutation may alter levels of lin-3 expression in the anchor cell. Using single molecule FISH, we therefore compared lin-3 transcript number in wild type, pept-1(lg601), lin-3(e1417) and lin-3(e1417); pept-1(lg601) animals  , which specifically reduces transcription in the anchor cell, are weaker than of lin-3(n378) (lin-3(mf75), a genetic background with no expression of lin-3 in the anchor cell, is insensitive to pept-1 RNAi, suggests that starvation effects on vulval induction can only manifest under minimal EGF-Ras-MAPK activity, corresponding to the situation where one or more lin-3 transcripts are present in the AC. One possible scenario is that the effect of such low doses of lin-3 on vulval induction can then become amplified under nutrient deprivation, e.g., at the level of the LIN-3 protein and its secretion, or at the level or downstream of the EGF receptor (LET-23)Given the above results, we next tested whether nutrient deprivation caused by the  animals . However3(e1417)  caused b-3(n378) . These rpry-1) may partly compensate for reduced EGF-Ras-MAPK activity during vulval induction, e.g., in let-23(rf) mutants. bar-1/β-Catenin  (lin-3(n378); bar-1(ga80) double mutants was not suppressed by starvation, implying that Wnt pathway activity acting via bar-1/β-Catenin is required for starvation suppression of lin-3(rf). However, it remained unclear to what extent starvation compromised induction vs. competence of vulval cells in bar-1(ga80) because in the study of bar-1(ga80) animals to starvation in late L1 vs. mid-L2 stage and found that mid-L2 starvation did not alter vulval induction whereas late L1 starvation caused strong hypoinduction, primarily due to Pn.p fusion, i.e., loss of competence of vulval precursor cells, including P5.p to P7.p (bar-1(ga80) fusion defects prior to vulval induction, so that this starvation treatment cannot be used to evaluate starvation effects on vulval inductive levels. In contrast, starvation at a later time point, at mid-L2 – covering the time period during which vulval induction is most sensitive to starvation – slightly, although not significantly, increased vulval induction of bar-1(ga80) animals (pept-1 RNAi treatment of bar-1(ga80) clearly and significantly increased vulval induction ; bar-1(ga80) double mutants to pept-1 RNAi treatment , or 48 hr of starvation at the mid-L2 stage , we observed no starvation suppression of this highly penetrant Vulvaless phenotype. This result suggests, similar to the results obtained for other highly penetrant Vulvaless mutants , that a very strong reduction in inductive signals (including contribution from BAR-1/Wnt) cannot be rescued by starvation effects. Nevertheless, given that vulval induction of the bar-1 null allele ga80 increases in response to starvation, we conclude that Wnt pathway/BAR-1 activity is not essential to mediate positive starvation effects on vulval inductive levels.In addition to the EGF-Ras-MAPK, starvation effects on vulval induction may also act through additional signaling pathways, in particular, the Wnt pathway. Previous results suggest that partial redundancy between Ras and Wnt pathway in vulval induction may allow for compensatory interplay when one of the two pathways is perturbed due to mutation . Gleason to P7.p . Late L1 animals . Yet, penduction , indicatlin-3(rf) and other EGF-Ras-MAPK mutations through the Wnt pathway  context (lin-3(rf) could be mediated in the same fashion. However, starvation did not alter vulval induction of lin-3(n378) in a bar-1(mu63) background (egl-30/Gαq (lin-3(n378)  . TherefoC. elegans vulval induction using mutant analysis and quantification of pathway activities allowed us to clarify how a specific environmental condition modulates C. elegans vulval induction (lin-3(rf) mutants, demonstrating that starvation exposure of >19h exerts a strong positive effect during the entire period of vulval induction, spanning from mid-L2 to early L3 stages. By testing various candidate mechanisms that could transduce and elicit observed starvation effects, we find that reduced activity of DAF-2 (Insulin) or DAF-7 (TGF-β) does not mimic lin-3(rf) starvation suppression. Instead, nutrient-deprived animals induced by mutation of the intestinal peptide transporter pept-1 or by the TOR pathway component rsks-1 (in a food-rich environment) strongly mimicked suppression of lin-3(rf) by external starvation. Moreover, we find that pept-1 RNAi is sufficient to increase both EGF-Ras-MAPK and Delta-Notch pathway activities in Pn.p cells. These results provide evidence for a link between nutrient deprivation, TOR-S6K and EGF-Ras-MAPK signaling during C. elegans vulval induction. It remains unclear how nutrient deprivation feeds into genetic pathways regulating vulval development, and we do not know whether this phenomenon represents a specific mechanism to fine-tune levels of vulval induction in response to the environment. In any case, the observed starvation modulation of C. elegans vulval induction provides an ideal study system to understand how organismal nutrient status can alter activities of major signaling pathways and penetrance of mutant phenotypes.Analysis of starvation sensitivity of nduction . We prespept-1 and rsks-1. Such slowing of development could potentially result in the accumulation of downstream effectors acting to promote vulval fates. Applied starvation treatments caused a strong delay in development: animals remained in the L2 stage throughout the 48-hr starvation period ; and pept-1 and rsks-1 mutants also showed important developmental delays relative to the wild type, with pept-1 showing a stronger developmental delay than rsks-1. Moreover, longer starvation caused increased suppression of lin-3(rf) phenotypes (starvation of 19h to 48 h) (lin-3(rf) was identical when starved for 48h to 120h)  background  . Importato 120h) , which ickground . Thus, ae.g., illustrated by the strong starvation suppression of lin-3(rf) mutations , implying starvation signal transduction via the sensory system (daf-2(rf) suppression of the Multivulva phenotype caused by let-60/Ras(gf) in food conditions was interpreted to suggest mimicing (of observed) starvation effects via reduced Insulin signaling (daf-2(rf); let-60(gf) responds to starvation. Our results reporting an overall positive effect of starvation on vulval induction suggest that sensory perception is not required for this effect to occur. Importantly, however, and in line with the results by DAF-2 activity reduces vulval induction in lin-3(rf) to similar extents in both food and starvation conditions, yet without abolishing starvation suppression of lin-3/egf(rf). Together, these different results suggest that starvation has both positive and negative effects on C. elegans vulval induction, with a positive starvation signal acting independently of external sensory inputs, likely mediated by nutrient sensing via TOR-S6K (this study), and a sensory-system-mediated negative starvation signal, likely involving DAF-2 signaling  remained very rare   . Therefo


Drosophila prothoracic gland (PG) was not a suitable strategy to disrupt gene expression, since Cas9 alone caused widespread lethality. The PG is widely used for studying endocrine gland function during animal development, but tools validating PG-specific RNAi phenotypes are lacking. Here, we present a collection of modular gateway-compatible CRISPR-Cas9 tools that allow precise modulation of target gene activity with temporal and spatial specificity. We also demonstrate that Cas9 fused to the progesterone ligand-binding domain can be used to activate gene expression via RU486. Using these approaches, we were able to avoid the lethality associated with simple GAL4-mediated overexpression of Cas9 in the PG. Given that the PG is a polytene tissue, we conclude that these tools work effectively in endoreplicating cells where Cas9 has to target multiple copies of the same locus. Our toolkit can be easily adapted for other tissues and can be used both for gain- and loss-of-function studies.Targeting gene function with spatial or temporal specificity is a key goal in molecular genetics. CRISPR-Cas9 has greatly facilitated this strategy, but some standard approaches are problematic. For instance, simple tissue-specific or global overexpression of Cas9 can cause significant lethality or developmental delays even in the absence of gRNAs. In particular, we found that Gal4-mediated expression of UAS-Cas9 in the  Drosophila, the standard techniques for conditionally altering gene function have been RNA interference (RNAi) to block or impair gene activity and the overexpression of cDNAs for gain-of-function studies. Most commonly, both RNAi and cDNA expression are temporally controlled via the Gal4-UAS system, resulting in a highly versatile set of tools. However, each of these commonly used components has its limitations and downsides. In particular, RNAi suffers from the frequent occurrence of off-targets, requiring rigorous validation, and often the expression of a target mRNA is only partially blocked. In addition, combining two or more RNAi transgenes to test for synthetic lethality or interaction of pathway components is cumbersome and exponentially increases the risk of non-specific effects. On the other hand, to achieve overexpression of a gene of interest, traditional cDNA overexpression requires the cloning of a full-length cDNA, which may be difficult and time-consuming. Further, in the case of alternatively spliced genes, one usually has to choose which isoform to use for the transgenic cDNA line, which may limit the conclusions that can be drawn from the experiment. It should also be noted that the use of Gal4 itself has its drawbacks. In particular, Gal4 may result in signal amplification due to its strong activation domain, and one has only limited control over how strongly a given cDNA is expressed. Further, UAS-regulated transgenes all show some degree of leakiness, depending on the tissue and developmental time, potentially confounding experimental outcomes , the German Cancer Research Center in Heidelberg (https://www.crisprflydesign.org/library/) and the National Institute of Genetics in Mishima, Japan (https://shigen.nig.ac.jp/fly/nigfly/).The recent discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the generation of guide RNA- (gRNA-) dependent Cas9 endonucleases has been quickly adapted by rd instar (L3) , which is part of the larval ring gland . The PG Drosophila, which we refer to as the general Gateway Cas9 (gG-Cas9), the PG-Cas9 and the PG-gRNA vector collections. These new tools have in common that they are not based on Gal4, but rather use enhancer regions to achieve tissue-specific expression of Cas9 or gRNAs. This greatly simplifies the genetics of tissue-specific CRISPR/Cas9, since one only requires a single cross to build the CRISPR/CAS9-gRNA combination, while the Gal4-UAS-based approach requires combining at least three transgenes. Importantly, we show that the lethality associated with Gal4-driven Cas9 can be prevented by several strategies, including tissue-specific expression of gRNAs coupled with ubiquitously expressed Cas9. To accomplish tissue-specific gRNA production, we took advantage of inserting ribozyme sequences, which demonstrates for the first time in Drosophila that ribozymes can be used to effectively release gRNAs from mRNAs. We also present the first fusion of Cas9 with the Ligand-binding domain of the human progesterone receptor and demonstrate that it is a highly effective tool for achieving both temporal and spatial control over Cas9-mediated gene activation. We evaluated the efficiency of each tool by targeting two well-studied genes acting in ecdysone biosynthesis, phantom and disembodied. Finally, we provide a general, gateway-based vector collection (gG-Cas9) that allows the quick generation of seven different Cas9-based vectors. These tissue-specific vectors enable the user to i) disrupt target genes of interest, ii) block the assembly of the transcription apparatus near the transcription start site or iii) upregulate the activity of a given gene.Here we present a collection of CRISPR tools designed for tissue-specific genome modification in 1v1PyX; P(carryP)attP40(II) (#25709), 1v1Py(nos-PhiC31/int.NLS)X; P(carryP)attP2(III) (#25710), E7/FM7cphm (#2208), 2/TM3 Sb1dib (#2776), UAS-Cas9.P2 (#58985), UAS-Cas9.P (#54594), UAS-Cas9.P (#54595), Act-Cas9 (#58590), spz5 P(OE.gRNA)attP40 (#67547), Alas P(OE.gRNA)attP40 (#68083), Coprox P(OE.gRNA)attP40 (#68124), FeCH P(OE.gRNA)attP40 (#78206), IRP1A P(OE.gRNA)attP40 (#68039), Nach P(OE.gRNA)attP40 (#67562) were obtained from the Bloomington Drosophila Stock Center.2cho2v1y (TBX-0004), 2cho2v1y; sco/CyO (TBX-0007), 2cho2v1/Yhs-hidy; Sp/CyO (TBX-0008), 2cho2v1y; Sp hs-hid/CyO (TBX-0009), 2cho2v1y; Pr Dr/TM6C, Sb Tb (TBX-0010) were obtained from the National Institute of Genetics of Japan (NIG).UAS-phm-RNAi (#108359), UAS-dib-RNAi (#101117) were obtained from the Vienna Drosophila Resource Center.Spok.DmCas9/TM3,Ser.GFP (Spok_DmC), Spok.HsCas9/CyO.GFP (Spok_HsC), Spok.Fok1-dCas9/CyO.GFP (Spok_dFC), Spok.dCas9-VP64b/TM6B,Hu,Tb (Spok_64bO), Spok.dCas9-GS-p65 (Spok_GSO), Spok.dCas9-VPR/TM6B,Hu,Tb (Spok_VPRO), Spok.dCas9/TM3,Ser.GFP (Spok_dI), 1v1;Py(pCFD3 no target gRNA)attP40 (pCFD3 control), 1v1;Py(pCFD5 no target gRNA)attP40 (pCFD5 control), 1v1;Py(PG1.gRNA no target gRNA)attP40 (pPG1.gRNA), 1v1;Py(PG2.gRNA no target gRNA)attP40 (pPG2.gRNA), 1v1;Py(PG3.gRNA no target gRNA)attP40 (pPG3.gRNA), 1v1;Py(pCFD5 phm.KO dgRNA)attP40 (dU6-phmgR1), 1v1;Py(pCFD5 phm gRNA SpokF9) (dU6-phmgR2), 1v1;Py(pCFD5 phm.5TSS -174 gRNA)attP40 (phm TSS-174), 1v1;Py(pCFD5 phm.5TSS -423 gRNA)attP40 (phm TSS-423), 1v1;Py(pCFD5 dib.KO dgRNA)attP40 (dU6-dibgR1),1v1;Py(pPG1.dib KO dgRNA)attP40 (pPG1-dibgR1), 1v1;Py(pCFD5 dib gRNA SpokF9) (dU6-dibgR2), 1v1;Py(pPG2.dib KO dgRNA)attP40 (pPG2-dibgR1), 1v1;Py(pPG3.dib KO dgRNA)attP40 (pPG-.dibgR1), 1v1;Py(pCFD5 dib.5TSS -110 gRNA)attP40 (dib TSS-110), 1v1;Py(pCFD5 dib.5TSS -482 gRNA)attP40 (dib TSS-482) were generated by our lab.1v1/SM5,CyOy, 1w*PyX; P(carryP)attP40(II) and 1w*Py(nos-PhiC31/int.NLS)X; P(carryP)attP2(III) were gifts from the BestGene Inc.phm22-Gal4, spok-Gal4/TM6 Tb and spok-Switch-Gal4 were kind gifts from Michael O’Connor’s lab. Stocks were maintained on a cornmeal diet unless otherwise specified.Drosophila Genomics Resources Center for generating spok_DmC. All PCR reactions were performed using NEB Q5 high fidelity DNA Polymerase (NEB M0491S) and purified by HighPrep PCR reagent from MagBio (AC-60005) following manufacturer’s protocol. All cloning steps were based on the Gibson reaction (We used the following plasmids that were available from Addgene: pAct:Cas9 (62209) for generating spok_HsC, pAct:dCas9-VP64 (78901) for generating spok_64aO, spok_64bO and spok_dI, pWalium20-10xUAS-3xFlag-dCas9-VPR (78897) for generating spok_VPRO, pP(ELAV-Geneswitch) (83957) for generating spok_GSO, pBPGUw (17575) for generating gG.Cas9 collection, pUC19 (50005), pCFD3 (49410), pCFD4 (49411) and pCFD5 (73914) . pAct:FoThe gG-Cas9 collection is based on the pBPGUw plasmid, which we modified to produce different Cas9 versions. This vector contains a Gateway Cassette, a synthetic core promoter and a Gal4-coding sequence (spok regulatory region amplified from pCRII-TOPO Spok plasmid (a kind gift from Michael O’Connor) via the Gibson reaction (Table S3). Constructs were then transformed into competent DH5α cells and validated by Sanger sequencing.To generate different PG-Cas9 constructs, we used PhiC31 vectors from the above-described gG-Cas9 collection. Vector backbones were amplified via PCR and fused with a 1.45kb fragment containing the spok regulatory region obtained from the CRII-TOPO Spok plasmid (a kind gift from Michael O’Connor). To ensure proper processing of the Pol II-derived transcript, we added either an HV or HDV ribozyme-containing region to the 3′ end, which was then amplified together with tRNA-gRNA duplexes (PG-specific gRNA plasmids were generated based on the pCFD5 plasmids that utilize tRNA- flanked gRNAs . We amplhttp://tools.flycrispr.molbio.wisc.edu/targetFinder/) and “Harvard CRISPR gRNA design tool” (http://www.flyrnai.org/crispr/). Optimal target sites were then confirmed by sequencing the loci from genomic DNA extracted from corresponding fly lines we used for plasmid injection. The pCFD5 and PG-gRNA plasmids were pre-digested with BbsI (NEB R3539S) and fused to appropriate gRNA-containing PCR fragments via the Gibson reaction, followed by Sanger sequencing. For PG2-gRNA, which only has a single BbsI cutting site, we used PG1 as a template to extend the scaffold for multiple gRNA sequences in the PG2 vector.Target gene sequences were obtained from FlyBase and analyzed for optimal target gRNA sites by selecting sequences that showed consensus between two programs, namely “CRISPR optimal target finder” X;P(carryP)attP40(II) or 1w*Py(nos-PhiC31/int.NLS)X;P(carryP)attP2(III) (for CRISPR/Cas9 constructs) or Bloomington #25709, #25710 (for gRNA constructs). Surviving adults were backcrossed to w1118 and screened for transformants.Plasmids were prepared by Qiagen plasmid midiprep (#12145) and eluted in nuclease-free water (Ambion Life Technologies AM9939) at a concentration of 500-600 ng/μl. Embryo injection was performed either at the University of Alberta or via GenetiVision Corporation following standard procedures (Drosophila Stock center formulation (https://bdsc.indiana.edu/information/recipes/bloomfood.html). For egg collections, flies were allowed to lay eggs for 3x 1-hour in order to reduce egg retention and minimize the presence of old embryos. Embryos were then collected in one-hour intervals, counted and transferred to vials containing appropriate media. Larval survival was scored at every stage. At least three independent crosses  were carried out per experimental condition. For 20OH-ecdysone (20E)-supplemented media, the final concentration was 0.33 mg/ml. To activate Cas9-GS (spokGS and spok_GSO), mifepristone/RU-486 (Sigma M8046) was used at a final concentration of 100 μg/mL, in line with other studies  is based on the Bloomington EcoRI and XbaI. Products were transformed into DH5α competent cells and colonies were randomly selected for Sanger sequencing.To analyze mutagenesis efficiency in the prothoracic gland (PG) conditional CRISPR, 15-20 ring glands were hand-dissected in collection buffer ) and incubated for 40 min at 37° before heat-inactivating proteinase K at 95° for 5 min. The target region was amplified from the extracted genomic PG DNA via PCR and cloned into the pUC19 vector (NewEngland Biolabs N3041S), which was pre-digested with Third instar larvae were collected 40-42 hr after the L2/L3 molt and brain-ring gland complexes (BRGC) were dissected and collected in 1xPBS. Samples were fixed in 1x PBS 4% formaldehyde (ThermoFisher 28906) for 20 min at room temperature (RT) before being washed in 1x PBS 0.3% Triton (Sigma T9284) (PBS3T) for 3x 10 min. Samples were blocked at RT for one hour in blocking solution ) and then incubated in primary antibody dilution buffer (antibody diluted in 1x PBS3T and 1% BSA) overnight at 4° with gentle shaking. Samples were then washed in 1x PBS3T for three times with 10 min each, incubated in secondary antibody dilution buffer for 1 hr at room temperature, washed in 1x PBS3T and 1:50,000 DAPI  for three times. Samples were mounted in Vectashield mounting medium (VECTH1000). Pictures were then taken using Nikon eclipse 80i confocal C2+ camera. To stain for Cas9, we used anti-CRISPR-Cas9 mouse monoclonal primary antibody (Abcam ab191468) at a ratio of 1:1000 and Goat anti-Mouse IgG H&L Alexa Fluor 555 secondary antibody (Abcam ab150114) was used at a ratio 1:2000.dib as well as -174 and -423 bp upstream of phm (relative to TSS), respectively. These conditions efficiently mimic in vivo conditions and allow physiological functions to be studied for up to 48 hr . 24 hr post transfection, 50 individual ring glands were collected per replicate, dissolved in Trizol, and stored at -80° for later qPCR analysis.BRGC were dissected from transgenic L3 larvae just after the L2/L3 molt, transferred to culture medium , and incubated at 25°. Larvae carried transgenes expressing gRNAs targeting -110 and -482 bp upstream of Larvae were raised on NutriFly media and staged at 42 hr after the L2/L3 molt. 50 individual ring glands were collected per replicate, dissolved in Trizol, and stored at -80° for later qPCR analysis. RNA was extracted using Qiagen RNeasy extraction kit (74106) and concentrations were measured via the RNA HS assay kit (Invitrogen Q32852) in a Qubit 2.0 (Invitrogen Q10210). Extracted samples were reverse- transcribed via the ABI High capacity cDNA synthesis kit (ThermoFisher 4368814). Synthesized cDNAs were used for qPCR  using the Luna Universal qPCR master mix (NEB M3003S). Samples were normalized to rp49 by calculating fold changes via the ΔΔCT method.https://doi.org/10.25387/g3.7075178.Fly strains and plasmids made by us are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are contained within the article, figures, and tables. Supplemental material available at Figshare: phm22-Gal4 , a widely used PG-specific Gal4 driver , which has overall lower expression levels compared to phm22> (Drosophila) and Cas9.P2 (codon-optimized for human cells) . Interestingly, ubiquitous expression of Cas9.P2 (act > Cas9.P2) caused no obvious lethality and the majority of the population reaches adulthood, while act > Cas9.C and act > Cas9.P were completely and partially lethal, respectively . We reasoned that the very high Cas9 expression levels that result from expressing UAS-Cas9 in combination with a strong PG-specific Gal4 drivers causes substantial cytotoxicity. Since the PG is responsible for producing ecdysteroids, high levels of Cas9 may interfere with ecdysteroid production and thus disrupt larval and pupal development. Similarly, PG-specific expression of dCas9.VPR had only 45% surviving adults, indicating that the toxicity is not necessarily linked to chromosomal breaks, as dCas9 does not cut DNA  fused to the VPR co-activator domain . Our ini4 driver . We theno phm22> . This obn cells) . UAS-Casn cells) . Unfortudulthood  and S1A. cut DNA . Taken tspok-Gal4GS, we activated Gal4 during the first (L1), second (L2), third (L3) or mid-third instar larval stages by transferring larvae to a RU486-supplemented diet. Temporal activation of Cas9 as late as early second instar still caused substantial lethality, while later stages (early and mid L3) displayed 60–70% survival. This might provide a suitable approach for studying gene function at later stages but would likely not be ideal for most genes expressed throughout larval development, such as the Halloween genes, which encode ecdysteroid-producing enzymes  system would resolve the problem. The GS system is based on a Gal4 DNA-binding domain that is fused to the human progesterone receptor ligand-binding domain and the activator domain from human p65 ( enzymes . However enzymes .enhancer-DmCas9 (en-DmC), enhancer-HsCas9 (en-HsC) and enhancer-FokI-dCas9 (en-dFC) function by generating double-strand breaks and deletions via site-directed Cleavage (C = cleavage), while enhancer-dCas9 (en-dI) acts via transcriptional interference. On the other hand, enhancer-dCas9-VP64 (en-64bO), enhancer-dCas9 GeneSwitch (en-GSO) and enhancer-dCas9-VPR (en-VPRO) are designed to overexpress target genes (O = overexpression) . More specifically, en-DmC is codon-optimized for Drosophila, while en-HsC is codon-optimized for humans . en-dFC is a fusion of dCas9 with the nuclease domain of FokI and designed to cut target DNA upon dimerization of the FokI nuclease domain (requires two gRNAs ∼15-25 bp apart). en-dI is designed for transcription interference (CRISPRi), where promoter-bound dCas9 will not cut DNA but rather sterically inhibit the proper formation of the pre-initiation complex. Taken together, this set of modified Gateway plasmids can be easily adapted to generate specific enhancer/Cas9 combinations, followed by PhiC31-mediated locus-specific transformation. In order to examine the efficiency of this system, we generated PG-specific versions of these vectors.We reasoned that omitting Gal4 altogether and instead opting for endogenous regulatory regions may result in lower but equally specific expression of Cas9, and thus reduce its toxicity. To accomplish this, we sought to generate a vector that would allow for quick insertion of tissue-specific enhancers. For this, we used the existing pBPGUw plasmid, a modular gateway-compatible Gal4 vector (spookier (spok) regulatory region , spok_HsC (human codon-optimized) and spok_dFC (FokI nuclease domain fused to fly codon-optimized dCas9). In order to test the efficiency for each approach, we generated transgenic lines carrying gRNAs against either phantom (phm) or disembodied (dib), two well characterized genes involved in ecdysteroid synthesis  in the coding region of phm or dib, we generated transgenic lines that carried at least two gRNAs (gR1dib and gR1phm), where the distance of the target sequence would not exceed 400 bp, although a distance of up to 750 bp has been reported to work as well  . Importantly, Cas9/gRNA animals were rescued to adulthood when reared on a diet supplemented with 20E, with typically 70–80% of the population developing into adults (Table S2), suggesting that the phenotypes resulted specifically from gene disruptions in phm and dib , in combination with two gRNAs, were highly efficient in generating deletions in the predicted region . When we crossed these four gRNA lines to flies carrying spok_dI transgenes, we observed developmental arrest during the L3 stage, which, in the case of dib, was comparable to what we had observed in phm22-Gal4 > dib-RNAi animals . This is a desirable approach for selectively targeting specific promoters of genes that harbor alternative promoters to repress specific mRNA isoforms. Alternatively, one could interfere with the expression of a gene for a defined duration, and then revert back to normal expression, thus studying dynamically expressed genes. We wanted to test whether PG-specific CRISPRi would work as efficiently as the other tools at our disposal. As candidate genes, we again chose  animals  and the  animals . In cont for dib . It is pll cases .dCas9-VP64 to dCas9-VP64b, so that it contained the same four nuclease-attenuating mutations as the dCas9-VPR construct.Previous approaches aimed at overexpressing a specific gene were based on the generation of transgenic lines that carry a cDNA, either driven by heat-shock promoters, nearby enhancers or the Gal4-UAS system . Later ispok_64aO, spok_64bO and spok_VPRO constructs by transfecting cultured brain-ring gland complexes (BRGC) that carried gRNA transgenes targeting either the phm or dib promoters upstream of the TSS, which were the same lines as used for CRISPRi. Since the plasmid-encoded Cas9 alleles were driven by the spok regulatory region, we reasoned that this approach should result in PG-specific Cas9 expression. Indeed, when we used ring gland-specific qPCR, spok_VPRO resulted in a 10- to 30-fold induction, while spok_64bO ranged from 5- to 15-fold upregulation. In contrast, the spok_64aO plasmid showed essentially no increased gene expression, suggestion that the two additional point mutations in spok_64bO (839A and N863A) are critical for induction  and that have comparatively flat expression profiles in the PG compared to phm and dib during larval development  system, where the Gal4 DNA-binding domain is fused to the human progesterone receptor-ligand-binding domain (hPR-LBD) and the p65 activation domain. The resulting chimeric Gal4 protein can only be activated in the presence of steroid mifepristone (RU486), typically provided in the diet. Therefore, we cloned a  and p65  and geneS system   restricting Cas9 expression to specific tissues or (ii) limiting the expression of gRNA to the tissue of interest. In the approaches outlined above, we employed PG-specific Cas9 expression. We therefore tested whether reversing gRNA and Cas9 expression patterns from tissue-specific to ubiquitous (and vice versa) was a viable strategy, because an existing line with ubiquitous Cas9 expression was reported to be homozygous viable (Bloomington stock #58590). A previous study described using UAS-driven multiplexed gRNA cloned into pCFD6 (Addgene 73915) to mediate tissue-specific gRNA expression in ng discs . In contng discs . This pUng discs . Howeverspok regulatory region and added sequences mediating hammerhead (HH) or Hepatitis delta virus (HDV) ribozyme function to induce self-cleavage and proper release of gRNAs (spok enhancer recruits Polymerase II (Pol II), resulting in mRNAs that will be subjected to 5′-capping and 3′-polyadenylation, which have the potential to interfere with proper gRNA maturation .In total, we generated four different PG-gRNA vectors, three of which are designed for generating DSBs (PG1-3), while one them (PG4) harbors an MS2 aptamer . For PG1gR1PG1-dib, gR1PG2-dib or gR1PG3-dib to Act-Cas9, we observed consistently 100% L3 arrest, similar to spok9>gR1pU6-dib animals that produced ubiquitous gRNA and PG-specific Cas9. In addition, supplementation with 20E efficiently rescued the L3 lethality, resulting in 72–82% normal-looking adults, indicating the specific disruption of the dib gene  (We showed here that PG-specific expression of Cas9 causes lethality that is independent of its nuclease activity. We have generated a series of strategies to solve this problem, which now allows somatic generation of DSBs, CRISPRi and CRISPRa. Generating tissue-specific gRNAs is also a viable strategy, provided ubiquitously expressed Cas9 levels are sufficiently low to avoid lethality. Since endoreplicating tissues harbor multiple copies of the same locus, Cas9 should be activated early enough to ensure efficient gene disruption. In our hands, somatic gRNAs work even better than RNAi lines, and appear to be highly specific, indicating that polytene tissues pose no issue for somatic CRISPR approaches. Even though we generated tools for PG-specific CRISPR, our tools can be used for any tissue of interest, polytene or not.