Blimp-1/PRDM1 and Hr3/RORß specify the blue-sensitive photoreceptor subtype in Drosophila by repressing the hippo pathway.

During terminal differentiation of the mammalian retina, transcription factors control binary cell fate decisions that generate functionally distinct subtypes of photoreceptor neurons. For instance, Otx2 and RORß activate the expression of the transcriptional repressor Blimp-1/PRDM1 that represses bipolar interneuron fate and promotes rod photoreceptor fate. Moreover, Otx2 and Crx promote expression of the nuclear receptor Nrl that promotes rod photoreceptor fate and represses cone photoreceptor fate. Mutations in these four transcription factors cause severe eye diseases such as retinitis pigmentosa. Here, we show that a post-mitotic binary fate decision in Drosophila color photoreceptor subtype specification requires ecdysone signaling and involves orthologs of these transcription factors: Drosophila Blimp-1/PRDM1 and Hr3/RORß promote blue-sensitive (Rh5) photoreceptor fate and repress green-sensitive (Rh6) photoreceptor fate through the transcriptional repression of warts/LATS, the nexus of the phylogenetically conserved Hippo tumor suppressor pathway. Moreover, we identify a novel interaction between Blimp-1 and warts, whereby Blimp-1 represses a warts intronic enhancer in blue-sensitive photoreceptors and thereby gives rise to specific expression of warts in green-sensitive photoreceptors. Together, these results reveal that conserved transcriptional regulators play key roles in terminal cell fate decisions in both the Drosophila and the mammalian retina, and the mechanistic insights further deepen our understanding of how Hippo pathway signaling is repurposed to control photoreceptor fates for Drosophila color vision.

Nicotine Has a Therapeutic Window of Effectiveness in a Drosophila melanogaster Model of Parkinson's Disease.

Strong epidemiological evidence and studies in models of Parkinson's disease (PD) suggest that nicotine may be therapeutically beneficial in PD patients. However, a number of clinical trials utilizing nicotine in PD patients have had mixed results, indicating that either nicotine is not beneficial in PD patients, or an important aspect of nicotine therapy was absent. We hypothesized that nicotine must be administered early in the adult fly life in order to have beneficial effects. We show that continuous early nicotine administration improves both climbing and flight deficiencies present in homozygous park 25 mutant PD model Drosophila melanogaster. Using a new climbing assay, we identify several climbing deficiencies in this PD model that are improved or rescued by continuous nicotine treatment. Amongst these benefits, it appears that nicotine improves the ability of the park 25 flies to descend the climbing vial by being able to climb down more. In support of our hypothesis, we show that in order for nicotine benefits on climbing and flight to happen, nicotine administration must occur in a discrete time frame following adult fly eclosure: within one day for climbing or five days for flight. This therapeutic window of nicotine administration in this PD model fly may help to explain the lack of efficacy of nicotine in human clinical trials.

The Blimp-1 transcription factor acts in non-neuronal cells to regulate terminal differentiation of the Drosophila eye.

The formation of a functional organ such as the eye requires specification of the correct cell types and their terminal differentiation into cells with the appropriate morphologies and functions. Here, we show that the zinc-finger transcription factor Blimp-1 acts in secondary and tertiary pigment cells in the Drosophila retina to promote the formation of a bi-convex corneal lens with normal refractive power, and in cone cells to enable complete extension of the photoreceptor rhabdomeres. Blimp-1 expression depends on the hormone ecdysone, and loss of ecdysone signaling causes similar differentiation defects. Timely termination of Blimp-1 expression is also important, as its overexpression in the eye has deleterious effects. Our transcriptomic analysis revealed that Blimp-1 regulates the expression of many structural and secreted proteins in the retina. Blimp-1 may function in part by repressing another transcription factor; Slow border cells is highly upregulated in the absence of Blimp-1, and its overexpression reproduces many of the effects of removing Blimp-1. This work provides insight into the transcriptional networks and cellular interactions that produce the structures necessary for visual function.

An altered microbiome in a Parkinson's disease model Drosophila melanogaster has a negative effect on development.

Parkinson's disease (PD) is the second most common neurodegenerative disease, besides Alzheimer's Disease, characterized by multiple symptoms, including the well-known motor dysfunctions. It is well-established that there are differences in the fecal microbiota composition between Parkinson's disease (PD) patients and control populations, but the mechanisms underlying these differences are not yet fully understood. To begin to close the gap between description and mechanism we studied the relationship between the microbiota and PD in a model organism, Drosophila melanogaster. First, fecal transfers were performed with a D. melanogaster model of PD that had a mutation in the parkin (park25) gene. Results indicate that the PD model feces had a negative effect on both pupation and eclosion in both control and park25 flies, with a greater effect in PD model flies. Analysis of the microbiota composition revealed differences between the control and park25 flies, consistent with many human studies. Conversely, gnotobiotic treatment of axenic embryos with feces-derived bacterial cultures did not affect eclosure. We speculate this result might be due to similarities in bacterial prevalence between mutant and control feces. Further, we confirmed a bacteria-potentiated impact on mutant and control fly phenotypes by measuring eclosure rate in park25 flies that were mono-associated with members of the fly microbiota. Both the fecal transfer and the mono-association results indicate a host genotype-microbiota interaction. Overall, this study concludes functional effects of the fly microbiota on PD model flies, providing support to the developing body of knowledge regarding the influence of the microbiota on PD.

Genome Sequence of Lactiplantibacillus plantarum DmPark25_157, a Bacterial Strain Isolated from Drosophila melanogaster.

We present the genome sequence of a bacterial strain isolated from park25 mutants of Drosophila melanogaster as part of efforts to better understand the microbial communities in D. melanogaster We isolated and sequenced a Lactiplantibacillus plantarum strain. We present a preliminary comparative analysis with a closely related strain.

Genome Sequence of Acetobacter tropicalis DmPark25_167, a Bacterium Isolated from a Drosophila melanogaster Genetic Model of Parkinson's Disease.

Here, we report the genome of Acetobacter tropicalis DmPark25_167, a bacterial strain isolated from a Drosophila melanogaster park25 mutant. The park25 mutant is an established genetic model of Parkinson's disease. DmPark25_167 has duplicated methionine metabolism and type IV secretion gene alleles compared with another strain of A. tropicalis.

Vulnerable Parkin Loss-of-Function Drosophila Dopaminergic Neurons Have Advanced Mitochondrial Aging, Mitochondrial Network Loss and Transiently Reduced Autophagosome Recruitment.

Selective degeneration of substantia nigra dopaminergic (DA) neurons is a hallmark pathology of familial Parkinson's disease (PD). While the mechanism of degeneration is elusive, abnormalities in mitochondrial function and turnover are strongly implicated. An Autosomal Recessive-Juvenile Parkinsonism (AR-JP) Drosophila melanogaster model exhibits DA neurodegeneration as well as aberrant mitochondrial dynamics and function. Disruptions in mitophagy have been observed in parkin loss-of-function models, and changes in mitochondrial respiration have been reported in patient fibroblasts. Whether loss of parkin causes selective DA neurodegeneration in vivo as a result of lost or decreased mitophagy is unknown. This study employs the use of fluorescent constructs expressed in Drosophila DA neurons that are functionally homologous to those of the mammalian substantia nigra. We provide evidence that degenerating DA neurons in parkin loss-of-function mutant flies have advanced mitochondrial aging, and that mitochondrial networks are fragmented and contain swollen organelles. We also found that mitophagy initiation is decreased in park (Drosophila parkin/PARK2 ortholog) homozygous mutants, but autophagosome formation is unaffected, and mitochondrial network volumes are decreased. As the fly ages, autophagosome recruitment becomes similar to control, while mitochondria continue to show signs of damage, and climbing deficits persist. Interestingly, aberrant mitochondrial morphology, aging and mitophagy initiation were not observed in DA neurons that do not degenerate. Our results suggest that parkin is important for mitochondrial homeostasis in vulnerable Drosophila DA neurons, and that loss of parkin-mediated mitophagy may play a role in degeneration of relevant DA neurons or motor deficits in this model.

Impaired climbing and flight behaviour in Drosophila melanogaster following carbon dioxide anaesthesia.

Laboratories that study Drosophila melanogaster or other insects commonly use carbon dioxide (CO2) anaesthesia for sorting or other work. Unfortunately, the use of CO2 has potential unwanted physiological effects, including altered respiratory and muscle physiology, which impact motor function behaviours. The effects of CO2 at different levels and exposure times were examined on the subsequent recovery of motor function as assessed by climbing and flight assays. With as little as a five minute exposure to 100% CO2, D. melanogaster exhibited climbing deficits up to 24 hours after exposure. Any exposure length over five minutes produced climbing deficits that lasted for days. Flight behaviour was also impaired following CO2 exposure. Overall, there was a positive correlation between CO2 exposure length and recovery time for both behaviours. Furthermore, exposure to as little as 65% CO2 affected the motor capability of D. melanogaster. These negative effects are due to both a CO2-specific mechanism and an anoxic effect. These results indicate a heretofore unconsidered impact of CO2 anaesthesia on subsequent behavioural tests revealing the importance of monitoring and accounting for CO2 exposure when performing physiological or behavioural studies in insects.

Computational tools for fitting the Hill equation to dose-response curves.

INTRODUCTION: Many biological response curves commonly assume a sigmoidal shape that can be approximated well by means of the 4-parameter nonlinear logistic equation, also called the Hill equation. However, estimation of the Hill equation parameters requires access to commercial software or the ability to write computer code. Here we present two user-friendly and freely available computer programs to fit the Hill equation - a Solver-based Microsoft Excel template and a stand-alone GUI-based "point and click" program, called HEPB. METHODS: Both computer programs use the iterative method to estimate two of the Hill equation parameters (EC50 and the Hill slope), while constraining the values of the other two parameters (the minimum and maximum asymptotes of the response variable) to fit the Hill equation to the data. In addition, HEPB draws the prediction band at a user-defined confidence level, and determines the EC50 value for each of the limits of this band to give boundary values that help objectively delineate sensitive, normal and resistant responses to the drug being tested. RESULTS: Both programs were tested by analyzing twelve datasets that varied widely in data values, sample size and slope, and were found to yield estimates of the Hill equation parameters that were essentially identical to those provided by commercial software such as GraphPad Prism and nls, the statistical package in the programming language R. DISCUSSION: The Excel template provides a means to estimate the parameters of the Hill equation and plot the regression line in a familiar Microsoft Office environment. HEPB, in addition to providing the above results, also computes the prediction band for the data at a user-defined level of confidence, and determines objective cut-off values to distinguish among response types (sensitive, normal and resistant). Both programs are found to yield estimated values that are essentially the same as those from standard software such as GraphPad Prism and the R-based nls. Furthermore, HEPB also has the option to simulate 500 response values based on the range of values of the dose variable in the original data and the fit of the Hill equation to that data.

Dissection and mounting of Drosophila pupal eye discs.

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Nicotine increases lifespan and rescues olfactory and motor deficits in a Drosophila model of Parkinson's disease.

Drosophila melanogaster is an attractive model of familial Parkinson's disease, as flies with loss-of-function mutations of the parkin gene exhibit many pathologies observed in PD patients. Progressive motor deficits found in homozygous parkin mutants seem to result from mitochondrial pathology that causes indirect flight muscle and dopaminergic neuronal degeneration [1,2]. We have found that heterozygous parkin mutants have decreased lifespan, generally progressive motor dysfunction and olfactory deficits compared to control flies, suggesting that mutation of this gene produces a dominant phenotype. Tobacco smokers are dose-dependently less likely to develop PD [3,4]; subsequent in vitro and in vivo studies show that nicotine is protective in models of sporadic PD [6]. Literature addressing the potential protection by nicotine in Parkin loss-of-function models spans limited concentrations and selected time points in the organism's lifespan. We have found that parkin heterozygotes have late-onset climbing and flying deficits as well as decreased viability and olfactory deficits that precede motor defects. While chronic nicotine exposure decreases lifespan and climbing and flying abilities in control flies, it can improve viability and flying capability as well as rescue climbing and olfactory deficits in parkin heterozygotes. Dopaminergic neurons are spared in the parkin heterozygote, perhaps because this phenotype is less severe than in the homozygous parkin mutants. Nicotine pretreatment may be protective in sporadic PD patients and models; however, timely diagnosis remains to be an obstacle. Our results suggest that nicotine also may be protective in familial PD patients, who can be easily identified before motor symptoms occur.

An airtight approach to the inebriometer: from construction to application with volatile anesthetics.

The mechanism by which inhaled anesthetics work is not fully understood, although they have been extensively used. Much research has been done showing the likelihood that there is more than one pathway or mechanism of action. A long-term goal of our laboratory is to decipher these mechanisms using Drosophila melanogaster, an excellent model organism for this purpose. In order to do this, we have modified and constructed an apparatus called the inebriometer to quantitatively analyze the response of flies to inhaled anesthetics. While the inebriometer is not new to the fly community, our updated design provides a relatively low-labor, high-throughput means for performing screens in search of genes involved in the anesthetic mechanism. Here we describe our construction of an airtight inebriometer that we have designed for this purpose. We also provide data that validates this apparatus and establishes a procedure for its use.

G-TRACE: rapid Gal4-based cell lineage analysis in Drosophila.

We combined Gal4-UAS and the FLP recombinase-FRT and fluorescent reporters to generate cell clones that provide spatial, temporal and genetic information about the origins of individual cells in Drosophila melanogaster. We named this combination the Gal4 technique for real-time and clonal expression (G-TRACE). The approach should allow for screening and the identification of real-time and lineage-traced expression patterns on a genomic scale.

Genomewide clonal analysis of lethal mutations in the Drosophila melanogaster eye: comparison of the X chromosome and autosomes.

Using a large consortium of undergraduate students in an organized program at the University of California, Los Angeles (UCLA), we have undertaken a functional genomic screen in the Drosophila eye. In addition to the educational value of discovery-based learning, this article presents the first comprehensive genomewide analysis of essential genes involved in eye development. The data reveal the surprising result that the X chromosome has almost twice the frequency of essential genes involved in eye development as that found on the autosomes.

Notch signaling controls germline stem cell niche formation in the Drosophila ovary.

Stem cells, which can self-renew and generate differentiated cells, have been shown to be controlled by surrounding microenvironments or niches in several adult tissues. However, it remains largely unknown what constitutes a functional niche and how niche formation is controlled. In the Drosophila ovary, germline stem cells (GSCs), which are adjacent to cap cells and two other cell types, have been shown to be maintained in the niche. In this study, we show that Notch signaling controls formation and maintenance of the GSC niche and that cap cells help determine the niche size in the Drosophila ovary. Expanded Notch activation causes the formation of more cap cells and bigger niches, which support more GSCs, whereas compromising Notch signaling during niche formation decreases the cap cell number and niche size and consequently the GSC number. Furthermore, the niches located away from their normal location can still sufficiently sustain GSC self-renewal by maintaining high local BMP signaling and repressing bam as in normal GSCs. Finally, loss of Notch function in adults results in rapid loss of the GSC niche, including cap cells and thus GSCs. Our results indicate that Notch signaling is important for formation and maintenance of the GSC niche, and that cap cells help determine niche size and function.

An efficient genetic screen in Drosophila to identify nuclear-encoded genes with mitochondrial function.

We conducted a screen for glossy-eye flies that fail to incorporate BrdU in the third larval instar eye disc but exhibit normal neuronal differentiation and isolated 23 complementation groups of mutants. These same phenotypes were previously seen in mutants for cytochrome c oxidase subunit Va. We have molecularly characterized six complementation groups and, surprisingly, each encodes a mitochondrial protein. Therefore, we believe our screen to be an efficient method for identifying genes with mitochondrial function.