Gain of function screen of PATs reveals an essential role of Hip14 in Drosophila host defense

The innate immune system is the first line of host defense against the invading pathogens in multicellular organisms,which is the key to eliminating pathogen infections,mitigating pathogen-induced inflammation,and activating adaptive immunity(Buchon et al.,2014;Liu and Cao 2016).

Efficient and Specific Modifications of the Drosophila Genome by Means of an Easy TALEN Strategy

Technology development has always been one of the forces driving breakthroughs in biomedical research. Since the time of Thomas Morgan, Drosophilists have, step by step, developed powerful genetic tools for manipulating and functionally dissecting the Drosophila genome, but room for improving these technologies and developing new techniques is still large, especially today as biologists start to study systematically the functional genomics of different model organisms, including humans, in a high-throughput manner. Here, we report, for the first time in Drosophila, a rapid, easy, and highly specific method for modifying the Drosophila genome at a very high efficiency by means of an improved transcription activator-like effector nuclease （TALEN） strategy. We took advantage of the very recently developed ＂unit assembly＂ strategy to assemble two pairs of specific TALENs designed to modify the yellow gene （on the sex chromosome） and a novel autosomal gene. The mRNAs of TALENs were subsequently injected into Drosophila embryos. From 31.2% of the injected Fo fertile flies, we detected inheritable modification involving the yellow gene. The entire process from construction of specific TALENs to detection of inheritable modifications can be accomplished within one month. The potential applications of this TALEN-mediated genome modification method in Drosophila are discussed.

Drosophila RecQ5 is required for efficient SSA repair and suppression of LOH in vivo

RecQ5 in mammalian cells has been suggested to suppress inappropriate homologous recombination.However,the specific pathway(s)in which it is involved and the underlining mechanism(s)remain poorly understood.We took advantage of genetic tools in Drosophila to investigate how Drosophila RecQ5(dRecQ5)functions in vivo in homologous recombination-mediated double strand break(DSB)repair.We generated null alleles of dRecQ5 using the targeted recombination technique.The mutant animals are homozygous viable,but with growth retardation during development.The mutants are sensitive to both exogenous DSB-inducing treatment,such as gamma-irradiation,and endogenously induced double strand breaks(DSBs)by I-Sce I endonuclease.In the absence of dRecQ5,single strand annealing(SSA)-mediated DSB repair is compromised with compensatory increases in either inter-homologous gene conversion,or non-homologous end joining(NHEJ)when inter-chromosomal homologous sequence is unavailable.Loss of function of dRecQ5 also leads to genome instability in loss of heterozygosity(LOH)assays.Together,our data demonstrate that dRecQ5 functions in SSA-mediated DSB repair to achieve its full efficiency and in suppression of LOH in Drosophila.

TALEN or Cas9-Rapid,Efficient and Specific Choices for Genome Modifications

Precise modifications of complex genomes at the single nucleotide level have been one of the big goals for scientists working in basic and applied genetics,including biotechnology,drug development,gene therapy and synthetic biology.However,the relevant techniques for making these manipulations in model organisms and human cells have been lagging behind the rapid high throughput studies in the post-genomic era with a bottleneck of low efficiency,time consuming and laborious manipulation,and off-targeting problems.Recent discoveries of TALEs（transcription activator-like effectors） coding system and CRISPR（clusters of regularly interspaced short palindromic repeats） immune system in bacteria have enabled the development of customized TALENs（transcription activator-like effector nucleases） and CRISPR/Cas9 to rapidly edit genomic DNA in a variety of cell types,including human cells,and different model organisms at a very high efficiency and specificity.In this review,we first briefly summarize the development and applications of TALENs and CRISPR/Cas9-mediated genome editing technologies;compare the advantages and constraints of each method;particularly,discuss the expected applications of both techniques in the field of site-specific genome modification and stem cell based gene therapy;finally, propose the future directions and perspectives for readers to make the choices.

A Systematic Phenotypic Screen of F-box Genes Through a Tissue-specific RNAi-based Approach in Drosophila

F-box proteins are components of the SCF （SkpA-Cullin 1-F-box） E3 ligase complexes, acting as the specificity-determinants in targeting substrate proteins for ubiquitination and degradation. In humans, at least 22 out of 75 F-box proteins have experimentally documented substrates, whereas in Drosophila 12 F-box proteins have been characterized with known substrates. To systematically investigate the genetic and molecular functions of F-box proteins in Drosophila, we performed a survey of the literature and databases. We identified 45 Drosophila genes that encode proteins containing at least one F-box domain. We collected publically available RNAi lines against these genes and used them in a tissue-specific RNAi-based phenotypic screen. Here, we present our systematic phenotypic dataset from the eye, the wing and the notum. This dataset is the first of its kind and represents a useful resource for future studies of the molecular and genetic functions of F-box genes in Drosophila. Our results show that, as expected, F-box genes in Drosophila have regulatory roles in a diverse array of processes including cell proliferation, cell growth, signal transduction, and cellular and animal survival.

HDAC6 regulates lipid droplet turnover in response to nutrient deprivation via p62-mediated selective autophagy

Autophagy has been evolved as one of the adaptive cellular processes in response to stresses such as nutrient deprivation. Various cellular cargos such as damaged organelles and protein aggregates can be selectively degraded through autophagy. Recently, the lipid storage organelle, lipid droplet(LD), has been reported to be the cargo of starvation-induced autophagy. However, it remains largely unknown how the autophagy machinery recognizes the LDs and whether it can selectively degrade LDs. In this study, we show that Drosophila histone deacetylase 6(dHDAC6), a key regulator of selective autophagy, is required for the LD turnover in the hepatocyte-like oenocytes in response to starvation. HDAC6 regulates LD turnover via p62/SQSTM1(sequestosome 1)-mediated aggresome formation, suggesting that the selective autophagy machinery is required for LD recognition and degradation. Furthermore, our results show that the loss of dHDAC6 causes steatosis in response to starvation. Our findings suggest that there is a potential link between selective autophagy and susceptible predisposition to lipid metabolism associated diseases in stress conditions.

The CRISPR/Cas9 Genome Editing Revolution

Genomes encode the genetic information that controls the development and physiological functions of all living organisms on our planet,and are therefore of central interest in all aspects of biomedical research.To understand the blueprint of life,scientists have long aimed to read and manipulate the genome using a rapidly expanding toolbox.To read the genome,novel state-of-the-art sequencing technologies have made it possible to sequence any single genome rapidly and cheaply.

Ecdysone and Insulin Signaling Play Essential Roles in Readjusting the Altered Body Size Caused by the dGPAT4 Mutation in Drosophila

Body size is one of the features that distinguish one species from another in the biological world. Animals have developed mechanisms to control their body size during normal development. However, how animals cope with genetic alterations and/or environmental stresses to develop into normal-sized adults remain poorly understood. The ability of the animals to develop into a normal-sized adult after the challenges of genetic alterations and/or environmental stresses reveals a robustness of body size control. Here we show that the mutation of dGPAT4, a de novo synthase of lysophosphatidic acid, is a genetic alteration that triggers such a robust response of the animals to body size challenges in Drosophila. Loss of dGPAT4 leads to a severe delay of development, slow growth and resultant small-sized animals during the larval stages, but results in normal-sized adult flies. The robust body size adjustment of the dGPAT4 mutant is likely achieved by corresponding changes in ecdysone and insulin signaling, which is also manifested by compromised food intake. Thus, we propose that a strategy has been evolved by the animals to reach final body size when challenged by genetic alterations, which requires the coordinated ecdysone and insulin signaling.

Anything impossible with CRISPR/Cas9？

Since the adaptation of the bacterial CRISPR/Cas9 system to eukaryotic cells,this＂magic＂tool has now been dramatically modified and applied to all kinds of biomedical research,clinics and agriculture（Jiao and Gao,2016）.The system was first reported to rapidly,specifically and effectively target genomic sequences in human cells（Cong et al.,2013;Jinek et al.,2013）.

Shaggy regulates tissue growth through Hippo pathway in Drosophila

The evolutionarily conserved Hippo pathway coordinates cell proliferation,differentiation and apoptosis to regulate organ growth and tumorigenesis.Hippo signaling activity is tightly controlled by various upstream signals including growth factors and cell polarity,but the full extent to which the pathway is regulated during development remains to be resolved.Here,we report the identification of Shaggy,the homolog of mammalian Gsk3β,as a novel regulator of the Hippo pathway in Drosophila.Our results show that Shaggy promotes the expression of Hippo target genes in a manner that is dependent on its kinase activity.Loss of Shaggy leads to Yorkie inhibition and downregulation of Hippo pathway target genes.Mechanistically,Shaggy acts upstream of the Hippo pathway and negatively regulates the abundance of the FERM domain containing adaptor protein Expanded.Our results reveal that Shaggy is functionally required for Crumbs/Slmb-mediated downregulation of Expanded in vivo,providing a potential molecular link between cellular architecture and the Hippo signaling pathway.

Advances in Drosophila gene targeting and related techniques

Functional biological research has benefited tremendously by analyses of the phenotypes of mutant organisms which can be generated through targeted mutation of genes.In Drosophila,compared with random mutagenesis methods gene targeting has gained its popularity because it can introduce any desired mutation into a gene of interest.However,applications of gene targeting have been limited because the targeting efficiency varies with different genes,and the time and labor of targeting procedure are intensive.Nevertheless,improvement of gene targeting and development of its variant technologies have received much attention of scientists.Here we review recent progress that has been made in expanding the applications of gene targeting,which include the ϕC31 integration system and zinc-finger nucleases induced gene targeting,and new strategies that generate more efficient and reliable gene targeting.