Michael Buszczak: Tracking the big game in stem cell identity

Buszczak is exploring the regulation of proteins that control stem cell identity in the fly

Searching Chromatin for Stem Cell Identity

Stem cells encapsulate the fundamental problem of metazoan biology in miniature: How do cells establish and maintain their fates? Increasing evidence indicates that stem cell chromatin activates proliferation genes and represses differentiation genes. Understanding how these configurations are stabilized by Polycomb group proteins will advance our understanding of embryonic development, tissue homeostasis, regeneration, aging, and oncogenesis

Systematic discovery of genetic modulation by Jumonji histone demethylases in Drosophila

Jumonji (JmjC) domain proteins influence gene expression and chromatin organization by way of histone demethylation, which provides a means to regulate the activity of genes across the genome. JmjC proteins have been associated with many human diseases including various cancers, developmental and neurological disorders, however, the shared biology and possible common contribution to organismal development and tissue homeostasis of all JmjC proteins remains unclear. Here, we systematically tested the function of all 13 Drosophila JmjC genes. Generation of molecularly defined null mutants revealed that loss of 8 out of 13 JmjC genes modify position effect variegation (PEV) phenotypes, consistent with their ascribed role in regulating chromatin organization. However, most JmjC genes do not critically regulate development, as 10 members are viable and fertile with no obvious developmental defects. Rather, we find that different JmjC mutants specifically alter the phenotypic outcomes in various sensitized genetic backgrounds. Our data demonstrate that, rather than controlling essential gene expression programs, Drosophila JmjC proteins generally act to “fine-tune” different biological processes

A Genetic Mosaic Screen Reveals Ecdysone-Responsive Genes Regulating Drosophila Oogenesis

Information about the supplemental materials can be found here: www.g3journal.org/lookup/suppl/doi:10.1534/g3.116.028951/-/DC1Multiple aspects of Drosophila oogenesis, including germline stem cell activity, germ cell differentiation, and follicle survival, are regulated by the steroid hormone ecdysone. While the transcriptional targets of ecdysone signaling during development have been studied extensively, targets in the ovary remain largely unknown. Early studies of salivary gland polytene chromosomes led to a model in which ecdysone stimulates a hierarchical transcriptional cascade, wherein a core group of ecdysone-sensitive transcription factors induce tissue-specific responses by activating secondary branches of transcriptional targets. More recently, genome-wide approaches have identified hundreds of putative ecdysone-responsive targets. Determining whether these putative targets represent bona fide targets in vivo, however, requires that they be tested via traditional mutant analysis in a cell-type specific fashion. To investigate the molecular mechanisms whereby ecdysone signaling regulates oogenesis, we used genetic mosaic analysis to screen putative ecdysone-responsive genes for novel roles in the control of the earliest steps of oogenesis. We identified a cohort of genes required for stem cell maintenance, stem and progenitor cell proliferation, and follicle encapsulation, growth, and survival. These genes encode transcription factors, chromatin modulators, and factors required for RNA transport, stability, and ribosome biogenesis, suggesting that ecdysone might control a wide range of molecular processes during oogenesis. Our results suggest that, although ecdysone target genes are known to have cell type-specific roles, many ecdysone response genes that control larval or pupal cell types at developmental transitions are used reiteratively in the adult ovary. These results provide novel insights into the molecular mechanisms by which ecdysone signaling controls oogenesis, laying new ground for future studies.ECU Open Access Publishing Support Fun

Influence of cyclin type and dose on mitotic entry and progression in the early Drosophila embryo

Cyclins are key cell cycle regulators, yet few analyses test their role in timing the events that they regulate. We used RNA interference and real-time visualization in embryos to define the events regulated by each of the three mitotic cyclins of Drosophila melanogaster, CycA, CycB, and CycB3. Each individual and pairwise knockdown results in distinct mitotic phenotypes. For example, mitosis without metaphase occurs upon knockdown of CycA and CycB. To separate the role of cyclin levels from the influences of cyclin type, we knocked down two cyclins and reduced the gene dose of the one remaining cyclin. This reduction did not prolong interphase but instead interrupted mitotic progression. Mitotic prophase chromosomes formed, centrosomes divided, and nuclei exited mitosis without executing later events. This prompt but curtailed mitosis shows that accumulation of cyclin function does not directly time mitotic entry in these early embryonic cycles and that cyclin function can be sufficient for some mitotic events although inadequate for others

Identification of novel filament-forming proteins in Saccharomyces cerevisiae and Drosophila melanogaster

A screen for GFP-tagged yeast proteins that can assemble into visible structures reveals four new filamentous structures in the cytoplasm formed by metabolic enzymes and translation factors

DNA replication times the cell cycle and contributes to the mid-blastula transition in Drosophila embryos

Deletion of S phase disrupts mitotic timing in maternally regulated cycles, but it doesn't alter the cell cycle once zygotic transcription has begun

The Drosophila blood brain barrier is maintained by GPCR-dependent dynamic actin structures

Formation of actin-rich structures along the lateral borders of subperineurial glial cells are induced and maintained by the G protein–coupled receptor Moody

Recombineering-mediated tagging of Drosophila genomic constructs for in vivo localization and acute protein inactivation

Studying gene function in the post-genome era requires methods to localize and inactivate proteins in a standardized fashion in model organisms. While genome-wide gene disruption and over-expression efforts are well on their way to vastly expand the repertoire of Drosophila tools, a complementary method to efficiently and quickly tag proteins expressed under endogenous control does not exist for fruit flies. Here, we describe the development of an efficient procedure to generate protein fusions at either terminus in an endogenous genomic context using recombineering. We demonstrate that the fluorescent protein tagged constructs, expressed under the proper control of regulatory elements, can rescue the respective mutations and enable the detection of proteins in vivo. Furthermore, we also adapted our method for use of the tetracysteine tag that tightly binds the fluorescent membrane-permeable FlAsH ligand. This technology allows us to acutely inactivate any tagged protein expressed under native control using fluorescein-assisted light inactivation and we provide proof of concept by demonstrating that acute loss of clathrin heavy chain function in the fly eye leads to synaptic transmission defects in photoreceptors. Our tagging technology is efficient and versatile, adaptable to any tag desired and paves the way to genome-wide gene tagging in Drosophila