Craig T. Woodard

The Drosophila βFTZ-F1 orphan nuclear receptor provides competence for stage-specific responses to the steroid hormone ecdysone

The acquisition of competence is a key mechanism for refining global signals to distinct spatial and temporal responses. The molecular basis of competence, however, remains poorly understood. Here, we show that the beta FTZ-F1 orphan nuclear receptor functions as a competence factor for stage-specific responses to the steroid hormone ecdysone during Drosophila metamorphosis. beta FTZ-F1 mutants pupariate normally in response to the late larval pulse of ecdysone but display defects in stage-specific responses to the subsequent ecdysone pulse in prepupae. The ecdysone-triggered genetic hierarchy that directs these developmental responses is severely attenuated in beta FTZ-F1 mutants, although ecdysone receptor expression is unaffected. This study define beta FTZ-F1 as an essential competence factor for stage-specific responses to a steroid signal and implicates interplay among nuclear receptors as a mechanism for achieving hormonal competence.

A molecular mechanism for the stage specificity of the Drosophila prepupal genetic response to ecdysone

Two successive pulses of ecdysone signal the ends of larval and prepupal development in Drosophila, inducing early and late puffs in the salivary gland polytene chromosomes. Early puff induction in prepupae is dependent on a preceding period of protein synthesis and low ecdysone concentration. We demonstrate here that the competence acquired during this interval can be provided by beta FTZ-F1, a nuclear hormone receptor superfamily member derived from the 75CD mid-prepupal puff. We show that beta FTZ-F1 represses its own transcription and is repressed by ecdysone, explaining its brief expression in mid-prepupae. We further show that ectopic beta FTZ-F1 expression leads to enhanced levels of ecdysone-induced BR-C, E74, and E75 early gene transcription and premature induction of the stage-specific 93F early puff and E93 transcription. These findings indicate that beta FTZ-F1 plays a central role in the prepupal genetic response to ecdysone and provide a molecular mechanism for stage-specific responses to steroid hormones.

ßFTZ-F1 and Matrix metalloproteinase 2 are required for fat-body remodeling in Drosophila

During metamorphosis, holometabolous insects eliminate obsolete larval tissues via programmed cell death. In contrast, tissues required for further development are retained and often remodeled to meet the needs of the adult fly. The larval fat body is involved in fueling metamorphosis, and thus it escapes cell death and is instead remodeled during prepupal development. The molecular mechanisms by which the fat body escapes programmed cell death have not yet been described, but it has been established that fat-body remodeling requires 20-hydroxyecdysone (20E) signaling. We have determined that 20E signaling is required within the fat body for the cell-shape changes and cell detachment that are characteristic of fat-body remodeling. We demonstrate that the nuclear hormone receptor ßFTZ-F1 is a key modulator of 20E hormonal induction of fat body remodeling and Matrix metalloproteinase 2 (MMP2) expression in the fat body. We show that induction of MMP2 expression in the fat body requires 20E signaling, and that MMP2 is necessary and sufficient to induce fat-body remodeling.

Orphan nuclear receptor βFTZ-F1 is required for muscle-driven morphogenetic events at the prepupal–pupal transition in Drosophila melanogaster

In Drosophila melanogaster, fluctuations in 20-hydroxyecdysone (ecdysone) titer coordinate gene expression, cell death, and morphogenesis during metamorphosis. Our previous studies have supported the hypothesis that betaFTZ-F1 (an orphan nuclear receptor) provides specific genes with the competence to be induced by ecdysone at the appropriate time, thus directing key developmental events at the prepupal-pupal transition. We are examining the role of betaFTZ-F1 in morphogenesis. We have made a detailed study of morphogenetic events during metamorphosis in control and betaFTZ-F1 mutant animals. We show that leg development in betaFTZ-F1 mutants proceeds normally until the prepupal-pupal transition, when final leg elongation is delayed by several hours and significantly reduced in the mutants. We also show that betaFTZ-F1 mutants fail to fully extend their wings and to shorten their bodies at the prepupal-pupal transition. We find that betaFTZ-F1 mutants are unable to properly perform the muscle contractions that drive these processes. Several defects can be rescued by subjecting the mutants to a drop in pressure during the normal time of the prepupal-pupal transition. Our findings indicate that betaFTZ-F1 directs the muscle contraction events that drive the major morphogenetic processes during the prepupal-pupal transition in Drosophila.

Bioinformatics and the Undergraduate Curriculum

Recent advances involving high-throughput techniques for data generation and analysis have made familiarity with basic bioinformatics concepts and programs a necessity in the biological sciences. Undergraduate students increasingly need training in methods related to finding and retrieving information stored in vast databases. The rapid rise of bioinformatics as a new discipline has challenged many colleges and universities to keep current with their curricula, often in the face of static or dwindling resources. On the plus side, many bioinformatics modules and related databases and software programs are free and accessible online, and interdisciplinary partnerships between existing faculty members and their support staff have proved advantageous in such efforts. We present examples of strategies and methods that have been successfully used to incorporate bioinformatics content into undergraduate curricula.

The Ftz‐F1 family: Orphan nuclear receptors regulated by novel protein–protein interactions

Publisher Summary This chapter discusses the Ftz-F1 (NR5A) family of nuclear receptors (NRs), which is a large group of proteins involved in a wide range of biological processes. Ftz-F1 family NRs can bind to DNA as monomers; in addition to the zinc finger DNA-binding motifs, these proteins contain a C-terminal extension of their DNA-binding domain (DBDs)—the Ftz-F1 box—that contributes to DNA-binding specificity. However, direct interactions with other DNA-binding proteins, including homeodomain-containing transcription factors, influence the function of Ftz-F1 family members in animals as diverse as flies and mice. Drosophila Ftz-F1, the founding member of this family, is required for embryonic patterning at early stages of development and for ecdysone-triggered metamorphosis at later stages of development. Studies in Drosophila revealed a novel protein–protein interaction between Ftz-F1 and the Hox protein Ftz. The Ftz/Ftz-F1 protein–protein interaction mimics the interaction between NRs and standard coactivators as Ftz contains an LXXLL (NR-box) motif. The interaction of Ftz with Ftz-F1 also promoted the evolution of ftz from a homeotic gene to a segmentation gene in modern day Drosophila. Other noncanonical interactions between Ftz-F1 family members and DNA-binding transcription factors may function similarly to restrict the cell-specific function of other Ftz-F1 family NRs during development. These findings are discussed in light of evidence that Ftz-F1 family NRs bind phospholipid ligands.

Expression of human amyloid precursor protein in the skeletal muscles of Drosophila results in age- and activity-dependent muscle weakness

BackgroundOne of the hallmarks of Alzheimers disease, and several other degenerative disorders such as Inclusion Body Myositis, is the abnormal accumulation of amyloid precursor protein (APP) and its proteolytic amyloid peptides. To better understand the pathological consequences of inappropriate APP expression on developing tissues, we generated transgenic flies that express wild-type human APP in the skeletal muscles, and then performed anatomical, electrophysiological, and behavioral analysis of the adults.ResultsWe observed that neither muscle development nor animal longevity was compromised in these transgenic animals. However, human APP expressing adults developed age-dependent defects in both climbing and flying. We could advance or retard the onset of symptoms by rearing animals in vials with different surface properties, suggesting that human APP expression-mediated behavioral defects are influenced by muscle activity. Muscles from transgenic animals did not display protein aggregates or structural abnormalities at the light or transmission electron microscopic levels. In agreement with genetic studies performed with developing mammalian myoblasts, we observed that co-expression of the ubiquitin E3 ligase Parkin could ameliorate human APP-induced defects.ConclusionsThese data suggest that: 1) ectopic expression of human APP in fruit flies leads to age- and activity-dependent behavioral defects without overt changes to muscle development or structure; 2) environmental influences can greatly alter the phenotypic consequences of human APP toxicity; and 3) genetic modifiers of APP-induced pathology can be identified and analyzed in this model.

Negotiating Peer Mentoring Roles in Undergraduate Research Lab Settings

Undergraduate research is viewed as an important catalyst for educational engagement and persistence, with an emphasis on the faculty mentoring relationship. Despite the common practice of having multi-tiered lab teams composed of newer undergraduates and more seasoned undergraduates serving as peer mentors, less is understood about the experience of peer mentors. Using the framework of legitimate peripheral participation, this study examined how peer mentors negotiated their roles in the lab. Nested case studies based on interviews with peer mentors, faculty members, and newer students illustrated how peer mentors establish credibility through prior lab experience and faculty-framed authority. Delegating supervision was an important component that helped newer students to accept the authority of the peer mentor. Implications for program development and future research involving peer mentoring are discussed.