Precise single base substitution in the shibire gene by CRISPR/Cas9-mediated homology directed repair in Bactrocera tryoni.

BACKGROUND: Pest eradication using the Sterile Insect Technique (SIT) involves high-density releases of sterilized males that mate with wild females and ultimately suppress the population. Sterilized females are not required for SIT and their removal or separation from males prior to release remains challenging. In order to develop genetic sexing strains (GSS), conditional traits such as temperature sensitive lethality are required. RESULTS: Here we introduce a known Drosophila melanogaster temperature sensitive embryonic lethal mutation into Bactrocera tryoni, a serious horticultural pest in Australia. A non-synonymous point mutation in the D. melanogaster gene shibire causes embryonic lethality at 29 °C and we successfully used CRISPR/Cas9 technology to recreate the orthologous shibire temperature sensitive-1 (shits1) mutation in B. tryoni. Genotypic analyses over three generations revealed that a high fitness cost was associated with the shits1 mutant allele and shits1 homozygotes were not viable at 21 °C, which is a more severe phenotype than that documented in D. melanogaster. CONCLUSIONS: We have demonstrated the first successful use of CRISPR/Cas9 to introduce precise single base substitutions in an endogenous gene via homology-directed repair in an agricultural pest insect and this technology can be used to trial other conditional mutations for the ultimate aim of generating genetic sexing strains for SIT.

Maintenance of Cell Fate by the Polycomb Group Gene Sex Combs Extra Enables a Partial Epithelial Mesenchymal Transition in Drosophila.

Epigenetic silencing by Polycomb group (PcG) complexes can promote epithelial-mesenchymal transition (EMT) and stemness and is associated with malignancy of solid cancers. Here we report a role for Drosophila PcG repression in a partial EMT event that occurs during wing disc eversion, an early event during metamorphosis. In a screen for genes required for eversion we identified the PcG genes Sexcombs extra (Sce) and Sexcombs midleg (Scm) Depletion of Sce or Scm resulted in internalized wings and thoracic clefts, and loss of Sce inhibited the EMT of the peripodial epithelium and basement membrane breakdown, ex vivo. Targeted DamID (TaDa) using Dam-Pol II showed that Sce knockdown caused a genomic transcriptional response consistent with a shift toward a more stable epithelial fate. Surprisingly only 17 genes were significantly upregulated in Sce-depleted cells, including Abd-B, abd-A, caudal, and nubbin Each of these loci were enriched for Dam-Pc binding. Of the four genes, only Abd-B was robustly upregulated in cells lacking Sce expression. RNAi knockdown of all four genes could partly suppress the Sce RNAi eversion phenotype, though Abd-B had the strongest effect. Our results suggest that in the absence of continued PcG repression peripodial cells express genes such as Abd-B, which promote epithelial state and thereby disrupt eversion. Our results emphasize the important role that PcG suppression can play in maintaining cell states required for morphogenetic events throughout development and suggest that PcG repression of Hox genes may affect epithelial traits that could contribute to metastasis.

Chromosomal instability causes sensitivity to protein folding stress and ATP depletion.

Aneuploidy -- having an unbalanced genome - is poorly tolerated at the cellular and organismal level. It gives rise to proteotoxic stress as well as a stereotypical oxidative shift which makes these cells sensitive to internal and environmental stresses. Using Drosophila as a model, we found that protein folding stress is exacerbated by redox stress that occurs in response to ongoing changes to ploidy (chromosomal instability, CIN). We also found that if de novo nucleotide synthesis is blocked, CIN cells are dependent on a high level of lysosome function to survive. Depletion of adenosine monophosphate (AMP) synthesis enzymes led to DNA damage in CIN cells, which showed elevated activity of the DNA repair enzyme activated poly(ADP ribose) polymerase (PARP). PARP activation causes depletion of its substrate, nicotinamide adenine dinucleotide (NAD+) and subsequent loss of Adenosine Tri-Phosphate (ATP), and we found that adding ATP or nicotinamide (a precursor in the synthesis of NAD+) could rescue the observed phenotypes. These findings provide ways to interpret, target and exploit aneuploidy, which has the potential to offer tumour-specific therapies.

Frazzled can act through distinct molecular pathways in epithelial cells to regulate motility, apical constriction, and localisation of E-Cadherin.

Netrin receptors of the DCC/NEO/UNC-40/Frazzled family have well established roles in cell migration and axon guidance but can also regulate epithelial features such as adhesion, polarity and adherens junction (AJ) stability. Previously, we have shown that overexpression of Drosophila Frazzled (Fra) in the peripodial epithelium (PE) inhibits wing disc eversion and also generates cellular protrusions typical of motile cells. Here, we tested whether the molecular pathways by which Fra inhibits eversion are distinct from those driving motility. We show that in disc proper (DP) epithelial cells Fra, in addition to inducing F-Actin rich protrusions, can affect localization of AJ components and columnar cell shape. We then show that these phenotypes have different requirements for the three conserved Fra cytoplasmic P-motifs and for downstream genes. The formation of protrusions required the P3 motif of Fra, as well as integrins (mys and mew), the Rac pathway (Rac1, wave and, arpc3) and myosin regulatory light chain (Sqh). In contrast, apico-basal cell shape change, which was accompanied by increased myosin phosphorylation, was critically dependent upon the P1 motif and was promoted by RhoGef2 but inhibited by Rac1. Fra also caused a loss of AJ proteins (DE-Cad and Arm) from basolateral regions of epithelial cells. This phenotype required all 3 P-motifs, and was dependent upon the polarity factor par6. par6 was not required for protrusions or cell shape change, but was required to block eversion suggesting that control of AJ components may underlie the ability of Fra to promote epithelial stability. The results imply that multiple molecular pathways act downstream of Fra in epithelial cells.

Phosphoenolpyruvate Carboxykinase Maintains Glycolysis-driven Growth in Drosophila Tumors.

Tumors frequently fail to pass on all their chromosomes correctly during cell division, and this chromosomal instability (CIN) causes irregular aneuploidy and oxidative stress in cancer cells. Our objective was to test knockdowns of metabolic enzymes in Drosophila to find interventions that could exploit the differences between normal and CIN cells to block CIN tumor growth without harming the host animal. We found that depleting by RNAi or feeding the host inhibitors against phosphoenolpyruvate carboxykinase (PEPCK) was able to block the growth of CIN tissue in a brat tumor explant model. Increasing NAD+ or oxidising cytoplasmic NADH was able to rescue the growth of PEPCK depleted tumors, suggesting a problem in clearing cytoplasmic NADH. Consistent with this, blocking the glycerol-3-phosphate shuttle blocked tumor growth, as well as lowering ROS levels. This work suggests that proliferating CIN cells are particularly vulnerable to inhibition of PEPCK, or its metabolic network, because of their compromised redox status.

Autophagy regulates the survival of cells with chromosomal instability.

Chromosomal instability (CIN) refers to genomic instability in which cells have gained or lost chromosomes or chromosomal fragments. A high level of CIN is common in solid tumours and is associated with cancer drug resistance and poor prognosis. The impact of CIN-induced stress and the resulting cellular responses are only just beginning to emerge. Using proliferating tissue in Drosophila as a model, we found that autophagy is activated in CIN cells and is necessary for their survival. Specifically, increasing the removal of defective mitochondria by mitophagy is able to lower levels of reactive oxygen species and the resultant cellular damage that is normally seen in CIN cells. In response to DNA damage, CIN is increased in a positive feedback loop, and we found that increasing autophagy by Tor depletion could decrease the level of CIN in proliferating cells. These findings underline the importance of autophagy control in the development of CIN tumours.

Chromosomal instability triggers cell death via local signalling through the innate immune receptor Toll.

Chromosomal instability (CIN) is a hallmark of cancer and has been implicated in cancer initiation, progression and the development of resistance to traditional cancer therapy. Here we identify a new property of CIN cells, showing that inducing CIN in proliferating Drosophila larval tissue leads to the activation of innate immune signalling in CIN cells. Manipulation of this immune pathway strongly affects the survival of CIN cells, primarily via JNK, which responds to both Toll and TNFα/Eiger. This pathway also activates Mmp1, which recruits hemocytes to the CIN tissue to provide local amplification of the immune response that is needed for effective elimination of CIN cells.

The organizer in evolution-gastrulation and organizer gene expression highlight the importance of Brachyury during development of the coral, Acropora millepora.

Organizer activity, once thought to be restricted to vertebrates, has ancient origins. However, among non-bilaterians, it has only been subjected to detailed investigation during embryonic development of the sea anemone, Nematostella vectensis. As a step toward establishing the extent to which findings in Nematostella can be generalized across the large and diverse phylum Cnidaria, we examined the expression of some key organizer and gastrulation genes during the embryonic development of the coral Acropora millepora. Although anemones and corals both belong to the cnidarian class Anthozoa, the two lineages diverged during the Cambrian and the morphological development of Acropora differs in several important respects from that of Nematostella. While the expression patterns of the key genes brachyury, bmp2/4, chordin, goosecoid and forkhead are broadly similar, developmental differences between the two species enable novel observations, and new interpretations of their significance. Specifically, brachyury expression during the flattened prawnchip stage before gastrulation, a developmental peculiarity of Acropora, leads us to suggest that it is the key gene demarcating ectoderm from endoderm in Acropora, and by implication in other cnidarians, whereas previous studies in Nematostella proposed that forkhead plays this role. Other novel observations include the transient expression of Acropora forkhead in scattered ectodermal cells shortly after gastrulation, and in the developing mesenterial filaments, with no corresponding expression reported in Nematostella. In addition, the expression patterns of goosecoid and bmp2/4 confirm the fundamental bilaterality of the Anthozoa.

Netrins and Frazzled/DCC promote the migration and mesenchymal to epithelial transition of Drosophila midgut cells.

Mesenchymal-epithelial transitions (METs) are important in both development and the growth of secondary tumours. Although the molecular basis for epithelial polarity is well studied, less is known about the cues that induce MET. Here we show that Netrins, well known as chemotropic guidance factors, provide a basal polarising cue during the Drosophila midgut MET. Both netrinA and netrinB are expressed in the visceral mesoderm, the substrate upon which midgut cells migrate, while their receptor frazzled (fra) is expressed in midgut cells. Netrins are required to polarise Fra to the basal surface, and Netrins and Fra undergo mutually-dependent endocytosis, with Fra subsequently trafficking to late endosomes. Mutations to fra and netrins affect both migration and MET but to different degrees. Loss of fra strongly delays migration, midgut cells fail to extend protrusions, and apico-basal polarisation of proteins and epithelium formation is inhibited. In netrin mutants, the migration phenotype is weaker and cells still extend protrusions. However, apico-basal polarisation of proteins, including Fra, and FActin is greatly disrupted and a monolayer fails to form. Delocalised accumulations of FActin are prevalent in netrin mutants but not fra mutants suggesting delocalised Fra may disrupt the MET. ßPS localisation is also affected in netrin mutants in that a basal gradient is reduced while localisation to the midgut/VM interface is increased. Since a similar effect is seen when endocytosis is inhibited, Netrin and Fra may regulate Integrin turnover. The results suggest Netrin-dependent basal polarisation of Fra is critical for the formation of an epithelium.

In vivo imaging and tracking of individual nanodiamonds in drosophila melanogaster embryos.

In this work, we incorporate and image individual fluorescent nanodiamonds in the powerful genetic model system Drosophila melanogaster. Fluorescence correlation spectroscopy and wide-field imaging techniques are applied to individual fluorescent nanodiamonds in blastoderm cells during stage 5 of development, up to a depth of 40 µm. The majority of nanodiamonds in the blastoderm cells during cellularization exhibit free diffusion with an average diffusion coefficient of (6 ± 3) × 10(-3) µm(2)/s, (mean ± SD). Driven motion in the blastoderm cells was also observed with an average velocity of 0.13 ± 0.10 µm/s (mean ± SD) µm/s and an average applied force of 0.07 ± 0.05 pN (mean ± SD). Nanodiamonds in the periplasm between the nuclei and yolk were also found to undergo free diffusion with a significantly larger diffusion coefficient of (63 ± 35) × 10(-3) µm(2)/s (mean ± SD). Driven motion in this region exhibited similar average velocities and applied forces compared to the blastoderm cells indicating the transport dynamics in the two cytoplasmic regions are analogous.

JNK signaling is needed to tolerate chromosomal instability.

Chromosomal instability (CIN), as a common feature of tumors, represents a potential therapeutic target if ways can be found to specifically cause apoptosis in unstably dividing cells. We have previously shown that if signaling through the JNK pathway is reduced, apoptosis is triggered in models of chromosomal instability induced by loss of the spindle checkpoint. Here we identify components upstream and downstream of JNK that are able to mediate this effect, and test the involvement of p53 and DNA damage in causing apoptosis when JNK signaling is reduced in CIN cells. We show that cell cycle progression timing has a strong effect on the apoptosis seen when JNK signaling is reduced in genetically unstable cells: a shortened G 2 phase enhances the apoptosis, while lengthening G 2 rescues the JNK-deficient CIN cell death phenotype. Our findings suggest that chromosomal instability represents a significant stress to dividing cells, and that without JNK signaling, cells undergo apoptosis because they lack a timely and effective response to DNA damage.

Netrin-dependent downregulation of Frazzled/DCC is required for the dissociation of the peripodial epithelium in Drosophila.

Netrins are secreted chemoattractants with roles in axon guidance, cell migration and epithelial plasticity. Netrin-1 also promotes the survival of metastasized cells by inhibiting the pro-apoptotic effects of its receptor Deleted in Colorectal Carcinoma (DCC). Here we report that Netrins can also regulate epithelial dissociation during Drosophila wing eversion. During eversion, peripodial epithelial cells lose apico-basal polarity and adherens junctions, and become migratory and invasive--a process similar to an epithelial-mesenchymal transition. Loss of netrinA inhibits the breakdown of cell-cell junctions, leading to eversion failure. In contrast, the Netrin receptor Frazzled blocks eversion when overexpressed, whereas frazzled RNAi accelerates eversion in vitro. In peripodial cells Frazzled is endocytosed, and undergoes NetA-dependent degradation, which is required for eversion. Finally, we provide evidence that Frazzled acts through the ERM-family protein Moesin to inhibit eversion. This mechanism may also help explain the role of Netrin and DCC in cancer metastasis.

C. elegans RNA-dependent RNA polymerases rrf-1 and ego-1 silence Drosophila transgenes by differing mechanisms.

Drosophila possesses the core gene silencing machinery but, like all insects, lacks the canonical RNA-dependent RNA polymerases (RdRps) that in C. elegans either trigger or enhance two major small RNA-dependent gene silencing pathways. Introduction of two different nematode RdRps into Drosophila showed them to be functional, resulting in differing silencing activities. While RRF-1 enhanced transitive dsRNA-dependent silencing, EGO-1 triggered dsRNA-independent silencing, specifically of transgenes. The strain w; da-Gal4; UAST-ego-1, constitutively expressing ego-1, is capable of silencing transgene including dsRNA hairpin upon a single cross, which created a powerful tool for research in Drosophila. In C. elegans, EGO-1 is involved in transcriptional gene silencing (TGS) of chromosome regions that are unpaired during meiosis. There was no opportunity for meiotic interactions involving EGO-1 in Drosophila that would explain the observed transgene silencing. Transgene DNA is, however, unpaired during the pairing of chromosomes in embryonic mitosis that is an unusual characteristic of Diptera, suggesting that in Drosophila, EGO-1 triggers transcriptional silencing of unpaired DNA during embryonic mitosis.

A screen for selective killing of cells with chromosomal instability induced by a spindle checkpoint defect.

BACKGROUND: The spindle assembly checkpoint is crucial for the maintenance of a stable chromosome number. Defects in the checkpoint lead to Chromosomal INstability (CIN), which is linked to the progression of tumors with poor clinical outcomes such as drug resistance and metastasis. As CIN is not found in normal cells, it offers a cancer-specific target for therapy, which may be particularly valuable because CIN is common in advanced tumours that are resistant to conventional therapy. PRINCIPAL FINDINGS: Here we identify genes that are required for the viability of cells with a CIN phenotype. We have used RNAi knockdown of the spindle assembly checkpoint to induce CIN in Drosophila and then screened the set of kinase and phosphatase genes by RNAi knockdown to identify those that induce apoptosis only in the CIN cells. Genes identified include those involved in JNK signaling pathways and mitotic cytoskeletal regulation. CONCLUSIONS/SIGNIFICANCE: The screen demonstrates that it is feasible to selectively kill cells with CIN induced by spindle checkpoint defects. It has identified candidates that are currently being pursued as cancer therapy targets (e.g. Nek2: NIMA related kinase 2), confirming that the screen is able to identify promising drug targets of clinical significance. In addition, several other candidates were identified that have no previous connection with mitosis or apoptosis. Further screening and detailed characterization of the candidates could potentially lead to the therapies that specifically target advanced cancers that exhibit CIN.

Snail-dependent repression of the RhoGEF pebble is required for gastrulation consistency in Drosophila melanogaster.

The Rho GTP exchange factor, Pebble (Pbl), long recognised as an essential activator of Rho during cytokinesis, also regulates mesoderm migration at gastrulation. Like other cell cycle components, pbl expression patterns broadly correlate with proliferative tissue. Surprisingly, in spite of its role in the early mesoderm, pbl is downregulated in the presumptive mesoderm before ventral furrow formation. Here, we show that this mesoderm-specific repression of pbl is dependent on the transcriptional repressor Snail (Sna). pbl repression was lost in sna mutants but was unaffected when Sna was ectopically expressed, showing that Sna is necessary, but not sufficient, for pbl repression. Using DamID, the first intron of pbl was identified as a Sna-binding region. Nine sites with the Sna-binding consensus motif CAGGT[GA] were identified in this intron. Mutating these to TAGGC[GA] abolished the ventral repression of pbl. Surprisingly, Sna-dependent repression of pbl was not essential for viability or fertility. Loss of repression did, however, increase the frequency of low-penetrance gastrulation defects. Consistent with this, expression of a pbl-GFP transgene in the presumptive mesoderm generated similar gastrulation defects. Finally, we show that a cluster of Snail-binding sites in the middle of the first intron of pbl orthologues is a conserved feature in the other 11 sequenced Drosophila species. We conclude that pbl levels are precisely regulated to ensure that there is enough protein available for its role in early mesoderm development but not so much as to inhibit the orderly progression of gastrulation.

Regulation of Drosophila mesoderm migration by phosphoinositides and the PH domain of the Rho GTP exchange factor Pebble.

The Drosophila RhoGEF Pebble (Pbl) is required for cytokinesis and migration of mesodermal cells. In a screen for genes that could suppress migration defects in pbl mutants we identified the phosphatidylinositol phosphate (PtdInsP) regulator pi5k59B. Genetic interaction tests with other PtdInsP regulators suggested that PtdIns(4,5)P2 levels are important for mesoderm migration when Pbl is depleted. Consistent with this, the leading front of migrating mesodermal cells was enriched for PtdIns(4,5)P2. Given that Pbl contains a Pleckstrin Homology (PH) domain, a known PtdInsP-binding motif, we examined PtdInsP-binding of Pbl and the importance of the PH domain for Pbl function. In vitro lipid blot assays showed that Pbl binds promiscuously to PtdInsPs, with binding strength associated with the degree of phosphorylation. Pbl was also able to bind lipid vesicles containing PtdIns(4,5)P2 but binding was strongly reduced upon deletion of the PH domain. Similarly, in vivo, loss of the PH domain prevented localisation of Pbl to the cell cortex and severely affected several aspects of early mesoderm development, including flattening of the invaginated tube onto the ectoderm, extension of protrusions, and dorsal migration to form a monolayer. Pbl lacking the PH domain could still localise to the cytokinetic furrow, however, and cytokinesis failure was reduced in pbl(ΔPH) mutants. Taken together, our results support a model in which interaction of the PH-domain of Pbl with PtdIns(4,5)P2 helps localise it to the plasma membrane which is important for mesoderm migration.

Imaging pluripotent cell migration in Drosophila.

Drosophila melanogaster offers a powerful system for the analysis of cell migration. In the embryo, -pluripotent cells of the mesodermal and endodermal primordia undergo epithelial-mesenchymal transitions and cell migration, while primordial germ cells migrate through an endodermal barrier to form the gonads. Visualisation of these migrations has traditionally been achieved by staining fixed embryos at different developmental stages or through live imaging of cells using tissue-specific expression of marker fluorescent proteins. More recently, photoactivatable fluorescence proteins have allowed the labelling of small groups of cells or single cells so that their migratory patterns and fate can be followed. By fusing the photoactivatable fluorescent protein to proteins that mark different subcellular components, it is now possible to visualise different aspects of the cells as they migrate. Here, we review previous studies of the migration of pluripotent embryonic cells and describe, in detail, methods for visualising these cells.

Expression of distinct RNAs from 3' untranslated regions.

The 3' untranslated regions (3'UTRs) of eukaryotic genes regulate mRNA stability, localization and translation. Here, we present evidence that large numbers of 3'UTRs in human, mouse and fly are also expressed separately from the associated protein-coding sequences to which they are normally linked, likely by post-transcriptional cleavage. Analysis of CAGE (capped analysis of gene expression), SAGE (serial analysis of gene expression) and cDNA libraries, as well as microarray expression profiles, demonstrate that the independent expression of 3'UTRs is a regulated and conserved genome-wide phenomenon. We characterize the expression of several 3'UTR-derived RNAs (uaRNAs) in detail in mouse embryos, showing by in situ hybridization that these transcripts are expressed in a cell- and subcellular-specific manner. Our results suggest that 3'UTR sequences can function not only in cis to regulate protein expression, but also intrinsically and independently in trans, likely as noncoding RNAs, a conclusion supported by a number of previous genetic studies. Our findings suggest novel functions for 3'UTRs, as well as caution in the use of 3'UTR sequence probes to analyze gene expression.

Polo kinase interacts with RacGAP50C and is required to localize the cytokinesis initiation complex.

The assembly and constriction of an actomyosin contractile ring in cytokinesis is dependent on the activation of Rho at the equatorial cortex by a complex, here termed the cytokinesis initiation complex, between a microtubule-associated kinesin-like protein (KLP), a member of the RacGAP family, and the RhoGEF Pebble. Recently, the activity of the mammalian Polo kinase ortholog Plk1 has been implicated in the formation of this complex. We show here that Polo kinase interacts directly with the cytokinesis initiation complex by binding RacGAP50C. We find that a new domain of Polo kinase, termed the intermediate domain, interacts directly with RacGAP50C and that Polo kinase is essential for localization of the KLP-RacGAP centralspindlin complex to the cell equator and spindle midzone. In the absence of Polo kinase, RacGAP50C and Pav-KLP fail to localize normally, instead decorating microtubules along their length. Our results indicate that Polo kinase directly binds the conserved cytokinesis initiation complex and is required to trigger centralspindlin localization as a first step in cytokinesis.

Cytokinesis proteins Tum and Pav have a nuclear role in Wnt regulation.

Wg/Wnt signals specify cell fates in both invertebrate and vertebrate embryos and maintain stem-cell populations in many adult tissues. Deregulation of the Wnt pathway can transform cells to a proliferative fate, leading to cancer. We have discovered that two Drosophila proteins that are crucial for cytokinesis have a second, largely independent, role in restricting activity of the Wnt pathway. The fly homolog of RacGAP1, Tumbleweed (Tum)/RacGAP50C, and its binding partner, the kinesin-like protein Pavarotti (Pav), negatively regulate Wnt activity in fly embryos and in cultured mammalian cells. Unlike many known regulators of the Wnt pathway, these molecules do not affect stabilization of Arm/beta-catenin (betacat), the principal effector molecule in Wnt signal transduction. Rather, they appear to act downstream of betacat stabilization to control target-gene transcription. Both Tum and Pav accumulate in the nuclei of interphase cells, a location that is spatially distinct from their cleavage-furrow localization during cytokinesis. We show that this nuclear localization is essential for their role in Wnt regulation. Thus, we have identified two modulators of the Wnt pathway that have shared functions in cell division, which hints at a possible link between cytokinesis and Wnt activity during tumorigenesis.