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An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in <em>Drosophila</em> by Genome Editing

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of ...

University of Maryland

Fragile X syndrome (FXS) is the most common cause of heritable intellectual disability and autism and affects ~1 in 4000 males and 1 in 8000 females. The discovery of effective treatments for FXS has been hampered by the lack of effective animal models and phenotypic readouts for drug screening. FXS ensues from the epigenetic silencing or loss-of-function mutation of the  () gene, which encodes an RNA binding protein that associates with and represses the translation of target mRNAs. We previously found that the activation of LIM kinase 1 (LIMK1) downstream of augmented synthesis of bone morphogenetic protein (BMP) type 2 receptor (BMPR2) promotes aberrant synaptic development in mouse and  models of FXS and that these molecular and cellular markers were correlated in patients with FXS. We report that larval locomotion is augmented in a  FXS model. Genetic or pharmacological intervention on the BMPR2-LIMK pathway ameliorated the synaptic abnormality and locomotion phenotypes of FXS larvae, as well as hyperactivity in an FXS mouse model. Our study demonstrates that (i) the BMPR2-LIMK pathway is a promising therapeutic target for FXS and (ii) the locomotion phenotype of FXS larvae is a quantitative functional readout for the neuromorphological phenotype associated with FXS and is amenable to the screening novel FXS therapeutics.

Hyperactive locomotion in a  model is a functional readout for the synaptic abnormalities underlying fragile X syndrome.

Fibroblast growth factors (FGF) are essential signaling proteins that regulate diverse cellular functions in developmental and metabolic processes. In Drosophila, the FGF homolog, branchless (bnl) is expressed in a dynamic and spatiotemporally restricted pattern to induce branching morphogenesis of the trachea, which expresses the Bnl-receptor, breathless (btl). Here we have developed a new strategy to determine bnl- expressing cells and study their interactions with the btl-expressing cells in the range of tissue patterning during Drosophila development. To enable targeted gene expression specifically in the bnl expressing cells, a new LexA based bnl enhancer trap line was generated using CRISPR/Cas9 based genome editing. Analyses of the spatiotemporal expression of the reporter in various embryonic stages, larval or adult tissues and in metabolic hypoxia, confirmed its target specificity and versatility. With this tool, new bnl expressing cells, their unique organization and functional interactions with the btl-expressing cells were uncovered in a larval tracheoblast niche in the leg imaginal discs, in larval photoreceptors of the developing retina, and in the embryonic central nervous system. The targeted expression system also facilitated live imaging of simultaneously labeled Bnl sources and tracheal cells, which revealed a unique morphogenetic movement of the embryonic bnl- source. Migration of bnl- expressing cells may create a dynamic spatiotemporal pattern of the signal source necessary for the directional growth of the tracheal branch. The genetic tool and the comprehensive profile of expression, organization, and activity of various types of bnl-expressing cells described in this study provided us with an important foundation for future research investigating the mechanisms underlying Bnl signaling in tissue morphogenesis.

Unique patterns of organization and migration of FGF-expressing cells during Drosophila morphogenesis.

Dinoflagellates are unicellular protists that feature a multitude of unusual nuclear features, including large genomes, packaging of DNA without histones, and multiple gene copies organized as tandem gene arrays. Furthermore, all dinoflagellate mRNAs experience trans-splicing with a common 22-nucleotide splice leader (SL) sequence. These features challenge some of the concepts and assumptions about the regulation of gene expression derived from work on model eukaryotes such as yeasts and mammals. Translational control in the dinoflagellates, based on extensive study of circadian bioluminescence and by more recent microarray and transcriptome analyses, is now understood to be a crucial element in regulating gene expression. A picture of the translation machinery of dinoflagellates is emerging from the recent availability of transcriptomes of multiple dinoflagellate species and the first complete genome sequences. The components comprising the translational control toolkit of dinoflagellates are beginning to take shape and are outlined here.

Sougata Roy

Department of Cell Biology and Molecular Genetics

University of Maryland

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Sougata Roy

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Cell Biology and Molecular Genetics

University of Maryland

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Department of Cell Biology and Molecular Genetics