Expression of an S phase-stabilized version of the CDK inhibitor Dacapo can alter endoreplication.

In developing organisms, divergence from the canonical cell division cycle is often necessary to ensure the proper growth, differentiation, and physiological function of a variety of tissues. An important example is endoreplication, in which endocycling cells alternate between G and S phase without intervening mitosis or cytokinesis, resulting in polyploidy. Although significantly different from the canonical cell cycle, endocycles use regulatory pathways that also function in diploid cells, particularly those involved in S phase entry and progression. A key S phase regulator is the Cyclin E-Cdk2 kinase, which must alternate between periods of high (S phase) and low (G phase) activity in order for endocycling cells to achieve repeated rounds of S phase and polyploidy. The mechanisms that drive these oscillations of Cyclin E-Cdk2 activity are not fully understood. Here, we show that the Drosophila Cyclin E-Cdk2 inhibitor Dacapo (Dap) is targeted for destruction during S phase via a PIP degron, contributing to oscillations of Dap protein accumulation during both mitotic cycles and endocycles. Expression of a PIP degron mutant Dap attenuates endocycle progression but does not obviously affect proliferating diploid cells. A mathematical model of the endocycle predicts that the rate of destruction of Dap during S phase modulates the endocycle by regulating the length of G phase. We propose from this model and our in vivo data that endo S phase-coupled destruction of Dap reduces the threshold of Cyclin E-Cdk2 activity necessary to trigger the subsequent G-S transition, thereby influencing endocycle oscillation frequency and the extent of polyploidy.

Using Drosophila Oogenesis in the Classroom to Increase Student Participation in Biomedical Research.

Students that participate in undergraduate research benefit in multiple ways, including improved learning outcomes, increased enthusiasm for science, technology, engineering, and mathematics (STEM) fields, and increased likelihood of continuation into a STEM career. These benefits are even more pronounced for students that are traditionally under-represented in STEM, although these students often face barriers to participation in traditional apprenticeship-style research experiences. Course-based undergraduate research experiences (CUREs) are a promising and increasingly popular approach to increase undergraduate participation in research in a way that is inclusive of all students. Here, we describe how Drosophila oogenesis can be used as the basis for CUREs in a wide variety of courses. We provide an overview of our own oogenesis-based CURE, as well as suggestions for how this CURE could be adapted to accommodate a variety of schedules, course sizes, and institution types. Our goal is to simplify the process for CURE implementation in the hopes that a greater number of instructors choose to implement a CURE in their own courses.

A directional recombination cloning system for restriction- and ligation-free construction of GFP, DsRed, and lacZ transgenic Drosophila reporters.

The fruit fly Drosophila is a leading model system for the study of transcriptional control by cis-regulatory elements, or enhancers. Here we present a rapid, high-efficiency system for directionally cloning PCR-amplified, PCR-mutated, or synthetic enhancer sequences into the Ganesh family of P element reporter constructs, which contain reporter genes encoding nuclear-localized eGFP, DsRed, or beta-galactosidase. This system, which is scalable for either small projects or high-throughput approaches, makes use of both TOPO and Gateway cloning technologies for directional, efficient cloning, without the need for restriction digestion or ligation reactions. It should be especially useful for those researchers who wish to test large numbers of putative enhancers, those who are undertaking detailed mutational analyses of enhancer sequences, or those who wish to avoid the difficulties sometimes encountered in traditional cloning strategies.

Sparkling insights into enhancer structure, function, and evolution.

This review focuses on a single cis-regulatory element: the sparkling eye enhancer of the Drosophila dPax2 gene. sparkling responds to Notch and EGFR signaling, along with other direct regulatory inputs, to drive gene expression that is restricted to cone cells of the developing fly retina. Functional, genetic, biochemical, evolutionary, and bioinformatic analyses have revealed surprising properties of sparkling, which may provide new insights into cis-regulatory logic and mechanisms of transcriptional activation. These properties include: a very high density of regulatory information and a correspondingly low "junk" content; an unexpectedly complex combinatorial code; tight functional constraints on enhancer organization, paradoxically coupled with high turnover of DNA sequence and binding site position; a requirement for weak binding of the transcription factor Su(H) to low-affinity sites in order to maintain a cell-type-specific response to Notch signaling; and multiple specialized regulatory sequences conferring functionally distinct activation activities, all of which are required in concert to achieve proper gene expression in vivo.

Characterization of null and hypomorphic alleles of the Drosophila l(2)dtl/cdt2 gene: Larval lethality and male fertility.

The Drosophila lethal(2)denticleless (l(2)dtl) gene was originally reported as essential for embryogenesis and formation of the rows of tiny hairs on the larval ventral cuticle known as denticle belts. It is now well-established that l(2)dtl (also called cdt2) encodes a subunit of a Cullin 4-based E3 ubiquitin ligase complex that targets a number of key cell cycle regulatory proteins, including p21, Cdt1, E2F1 and Set8, to prevent replication defects and maintain cell cycle control. To investigate the role of l(2)dtl/cdt2 during development, we characterized existing l(2)dtl/cdt2 mutants and generated new deletion alleles, using P-element excision mutagenesis. Surprisingly, homozygous l(2)dtl/cdt2 mutant embryos developed beyond embryogenesis, had intact denticle belts, and lacked an observable embryonic replication defect. These mutants died during larval stages, affirming that loss of l(2)dtl/cdt2 function is lethal. Our data show that L(2)dtl/Cdt2 is maternally deposited, remains nuclear throughout the cell cycle, and has a previously unreported, elevated expression in the developing gonads. We also find that E2f1 regulates l(2)dtl/cdt2 expression during embryogenesis, possibly via several highly conserved putative E2f1 binding sites near the l(2)dtl/cdt2 promoter. Finally, hypomorphic allele combinations of the l(2)dtl/cdt2 gene result in a novel phenotype: viable, low-fertility males. We conclude that "denticleless" is a misnomer, but that l(2)dtl/cdt2 is an essential gene for Drosophila development.

Rapid evolutionary rewiring of a structurally constrained eye enhancer.

BACKGROUND: Enhancers are genomic cis-regulatory sequences that integrate spatiotemporal signals to control gene expression. Enhancer activity depends on the combination of bound transcription factors as well as-in some cases-the arrangement and spacing of binding sites for these factors. Here, we examine evolutionary changes to the sequence and structure of sparkling, a Notch/EGFR/Runx-regulated enhancer that activates the dPax2 gene in cone cells of the developing Drosophila eye. RESULTS: Despite functional and structural constraints on its sequence, sparkling has undergone major reorganization in its recent evolutionary history. Our data suggest that the relative strengths of the various regulatory inputs into sparkling change rapidly over evolutionary time, such that reduced input from some factors is compensated by increased input from different regulators. These gains and losses are at least partly responsible for the changes in enhancer structure that we observe. Furthermore, stereotypical spatial relationships between certain binding sites ("grammar elements") can be identified in all sparkling orthologs-although the sites themselves are often recently derived. We also find that low binding affinity for the Notch-regulated transcription factor Su(H), a conserved property of sparkling, is required to prevent ectopic responses to Notch in noncone cells. CONCLUSIONS: Rapid DNA sequence turnover does not imply either the absence of critical cis-regulatory information or the absence of structural rules. Our findings demonstrate that even a severely constrained cis-regulatory sequence can be significantly rewired over a short evolutionary timescale.

Structural rules and complex regulatory circuitry constrain expression of a Notch- and EGFR-regulated eye enhancer.

Enhancers integrate spatiotemporal information to generate precise patterns of gene expression. How complex is the regulatory logic of a typical developmental enhancer, and how important is its internal organization? Here, we examine in detail the structure and function of sparkling, a Notch- and EGFR/MAPK-regulated, cone cell-specific enhancer of the Drosophila Pax2 gene, in vivo. In addition to its 12 previously identified protein-binding sites, sparkling is densely populated with previously unmapped regulatory sequences, which interact in complex ways to control gene expression. One segment is essential for activation at a distance, yet dispensable for other activation functions and for cell type patterning. Unexpectedly, rearranging sparkling's regulatory sites converts it into a robust photoreceptor-specific enhancer. Our results show that a single combination of regulatory inputs can encode multiple outputs, and suggest that the enhancer's organization determines the correct expression pattern by facilitating certain short-range regulatory interactions at the expense of others.