Biology subjects

Beitel, G. J.

Publications and source records attributed to Beitel, G. J..

3 recordsLinked to original sources

bioRxiv2020

Adaptable and Efficient Genome Editing by sgRNA-Cas9 Protein Co-injection into Drosophila

Genome editing via homology-directed repair (HDR) has made possible precise and deliberate modifications to gene sequences. CRISPR/Cas9-mediated HDR is the simplest means to carry this out. However, technical challenges remain to improve efficiency and broaden applicability to any genetic background of Drosophila melanogaster as well as to other Drosophila species. To address these issues, we developed a two-stage marker-assisted strategy in which embryos are injected with RNPs and pre-screened using T7EI. Using sgRNA in complex with recombinant Cas9 protein, we assayed each sgRNA for genome-cutting efficiency. We then conducted HDR using sgRNAs that efficiently cut target genes and the application of a transformation marker that generates RNAi against eyes absent. This allows for screening based on eye morphology rather than color. These new tools can be used to make a single change or a series of allelic substitutions in a region of interest, or to create additional genetic tools such as balancer chromosomes.

genetics↗

bioRxiv2018

The Caspase-3 homolog DrICE regulates endocytic trafficking during Drosophila tracheal morphogenesis

Although well known for its role in apoptosis, the executioner caspase DrICE has a non-apoptotic function that is required for elongation of the epithelial tubes of the Drosophila tracheal system. Here, we show that DrICE acts downstream of the Hippo Network to regulate endocytic trafficking of at least four cell polarity, cell junction and apical extracellular matrix proteins involved in tracheal tube size control: Crumbs, Uninflatable, Kune-Kune and Serpentine. We further show that tracheal cells are competent to undergo apoptosis, even though developmentally-regulated DrICE function rarely kills tracheal cells. Our results reveal a novel developmental role for caspases, a previously unidentified pool of DrICE that colocalizes with Clathrin, and a mechanism by which the Hippo Network controls endocytic trafficking. Given published reports of in vitro regulation of endocytosis by mammalian caspases during apoptosis, we propose that caspase-mediated regulation of endocytic trafficking is an evolutionarily conserved function of caspases that can be deployed during morphogenesis.

cell biology↗

bioRxiv2018

QuBiT: A quantitative tool for epithelial tubes reveals cell dynamics and unexpected patterns of organization during Drosophila tracheal morphogenesis

Biological tubes are essential for animal survival, and their functions are critically dependent on tube shape. Analyzing the contributions of cell shape and organization to the morphogenesis of small tubes has been hampered by the limitations of existing programs in quantifying cell geometry on highly curved tubular surfaces and calculating tube-specific parameters. We therefore developed QuBiT (Quantitative Tool for Biological Tubes) and used it to analyze morphogenesis during embryonic Drosophila tracheal (airway) development. We find that there are previously unknown anterior-to-posterior (A-P) gradients of cell orientation and aspect ratio, and that there is periodicity in the organization of cells in the main tube. Furthermore, cell intercalation during development dampens an A-P gradient of the number of the number of cells per cross-section of the tube, but these intercalation events do not change the patterns of cell connectivity. These unexpected findings demonstrate the importance of a computational tool for analyzing the morphogenesis of small diameter biological tubes.

cell biology↗