Activated Armadillo/β-Catenin Does Not Play a General Role in Cell Migration and Process Extension in Drosophila

Human beta-catenin and its fly homolog Armadillo are best known for their roles in cadherin-based cell-cell adhesion and in transduction of Wingless/Wnt signals. It has been hypothesized that beta-catenin may also regulate cell migration and cell shape changes, possibly by regulating the microtubule cytoskeleton via interactions with APC. This hypothesis was based on experiments in which a hyperstable mutant form of beta-catenin was expressed in MDCK cells, where it altered their migratory properties and their ability to send out long cellular processes. We tested the generality of this hypothesis in vivo in Drosophila. We utilized three model systems in which cell migration and/or process extension are known to play key roles during development: the migration of the border cells during oogenesis, the extension of axons in the nervous system, and the migration and cell process extension of tracheal cells. In all cases, cells expressing activated Armadillo were able to migrate and extend cell processes essentially normally. The one alteration from normal involved an apparent cell fate change in certain tracheal cells. These results suggest that only certain cells are affected by activation of Armadillo/beta-catenin, and that Armadillo/beta-catenin does not play a general role in inhibiting cell migration or process extension

A Functionally Conserved Gene Regulatory Network Module Governing Olfactory Neuron Diversity

Sensory neuron diversity is required for organisms to decipher complex environmental cues. In Drosophila, the olfactory environment is detected by 50 different olfactory receptor neuron (ORN) classes that are clustered in combinations within distinct sensilla subtypes. Each sensilla subtype houses stereotypically clustered 1–4 ORN identities that arise through asymmetric divisions from a single multipotent sensory organ precursor (SOP). How each class of SOPs acquires a unique differentiation potential that accounts for ORN diversity is unknown. Previously, we reported a critical component of SOP diversification program, Rotund (Rn), increases ORN diversity by generating novel developmental trajectories from existing precursors within each independent sensilla type lineages. Here, we show that Rn, along with BarH1/H2 (Bar), Bric-à-brac (Bab), Apterous (Ap) and Dachshund (Dac), constitutes a transcription factor (TF) network that patterns the developing olfactory tissue. This network was previously shown to pattern the segmentation of the leg, which suggests that this network is functionally conserved. In antennal imaginal discs, precursors with diverse ORN differentiation potentials are selected from concentric rings defined by unique combinations of these TFs along the proximodistal axis of the developing antennal disc. The combinatorial code that demarcates each precursor field is set up by cross-regulatory interactions among different factors within the network. Modifications of this network lead to predictable changes in the diversity of sensilla subtypes and ORN pools. In light of our data, we propose a molecular map that defines each unique SOP fate. Our results highlight the importance of the early prepatterning gene regulatory network as a modulator of SOP and terminally differentiated ORN diversity. Finally, our model illustrates how conserved developmental strategies are used to generate neuronal diversity

Patterns of transcriptional parallelism and variation in the developing olfactory system of Drosophila species

Organisms have evolved strikingly parallel phenotypes in response to similar selection pressures suggesting that there may be shared constraints limiting the possible evolutionary trajectories. For example, the behavioral adaptation of specialist Drosophila species to specific host plants can exhibit parallel changes in their adult olfactory neuroanatomy. We investigated the genetic basis of these parallel changes by comparing gene expression during the development of the olfactory system of two specialist Drosophila species to that of four other generalist species. Our results suggest that the parallelism observed in the adult olfactory neuroanatomy of ecological specialists extends more broadly to their developmental antennal expression profiles, and to the transcription factor combinations specifying olfactory receptor neuron (ORN) fates. Additionally, comparing general patterns of variation for the antennal transcriptional profiles in the adult and developing olfactory system of the six species suggest the possibility that specific, non-random components of the developmental programs underlying the Drosophila olfactory system harbor a disproportionate amount of interspecies variation. Further examination of these developmental components may be able to inform a deeper understanding of how traits evolve

Hybrid Neurons in a MicroRNA Mutant Are Putative Evolutionary Intermediates in Insect CO_2 Sensory Systems

Carbon dioxide (CO_2) elicits different olfactory behaviors across species. In Drosophila, neurons that detect CO_2 are located in the antenna, form connections in a ventral glomerulus in the antennal lobe, and mediate avoidance. By contrast, in the mosquito these neurons are in the maxillary palps (MPs), connect to medial sites, and promote attraction. We found in Drosophila that loss of a microRNA, miR-279, leads to formation of CO_2 neurons in the MPs. miR-279 acts through down-regulation of the transcription factor Nerfin-1. The ectopic neurons are hybrid cells. They express CO_2 receptors and form connections characteristic of CO_2 neurons, while exhibiting wiring and receptor characteristics of MP olfactory receptor neurons (ORNs). We propose that this hybrid ORN reveals a cellular intermediate in the evolution of species-specific behaviors elicited by CO_2

Cell cycle: Flies teach an old dogma new tricks

AbstractE2F transcription factors are thought to influence the G1–S cell-cycle transition by controlling expression of genes required for growth and DNA synthesis. But emerging evidence suggests E2F complexes can control the cell cycle independently of transcription by directly regulating DNA replication origin usage during S phase

Transcriptional Repressor Functions of Drosophila E2F1 and E2F2 Cooperate To Inhibit Genomic DNA Synthesis in Ovarian Follicle Cells

Individual members of the E2F/DP protein family control cell cycle progression by acting predominantly as an activator or repressor of transcription. In Drosophila melanogaster the E2f1, E2f2, Dp, and Rbf1 genes all contribute to replication control in ovarian follicle cells, which become 16C polyploid and subsequently undergo chorion gene amplification late in oogenesis. Mutation of E2f2, Dp, or Rbf1 causes ectopic DNA replication throughout the follicle cell genome during gene amplification cycles. Here we show by both reverse transcription-PCR and DNA microarray analysis that the transcripts of prereplication complex (pre-RC) genes are elevated compared to the wild type in E2f2, Dp, and Rbf1 mutant follicle cells. For some genes the magnitude of this transcriptional derepression is greater in Rbf1 than in E2f2 mutants. These differences correlate with differences in the magnitude of the replication defects in follicle cells, which attain an inappropriate 32C DNA content in both Rbf1 and Dp mutants but not in E2f2 mutants. The ectopic genomic replication of E2f2 mutant follicle cells can be suppressed by reducing the Orc2, Orc5, or Mcm2 gene dose by half, indicating that small changes in pre-RC gene expression can affect DNA synthesis in these cells. We conclude that RBF1 forms complexes with both E2F1/DP and E2F2/DP that cooperate to repress the expression of pre-RC genes, which helps confine DNA synthesis to sites of gene amplification. In contrast, E2F1 and E2F2 repressors function redundantly for some genes in the embryo. Thus, the relative functional contributions of E2F1 and E2F2 to gene expression and cell cycle control depends on the developmental context

Chromatin Modulatory Proteins and Olfactory Receptor Signaling in the Refinement and Maintenance of Fruitless Expression in Olfactory Receptor Neurons.

During development, sensory neurons must choose identities that allow them to detect specific signals and connect with appropriate target neurons. Ultimately, these sensory neurons will successfully integrate into appropriate neural circuits to generate defined motor outputs, or behavior. This integration requires a developmental coordination between the identity of the neuron and the identity of the circuit. The mechanisms that underlie this coordination are currently unknown. Here, we describe two modes of regulation that coordinate the sensory identities of Drosophila melanogaster olfactory receptor neurons (ORNs) involved in sex-specific behaviors with the sex-specific behavioral circuit identity marker fruitless (fru). The first mode involves a developmental program that coordinately restricts to appropriate ORNs the expression of fru and two olfactory receptors (Or47b and Ir84a) involved in sex-specific behaviors. This regulation requires the chromatin modulatory protein Alhambra (Alh). The second mode relies on the signaling from the olfactory receptors through CamK and histone acetyl transferase p300/CBP to maintain ORN-specific fru expression. Our results highlight two feed-forward regulatory mechanisms with both developmentally hardwired and olfactory receptor activity-dependent components that establish and maintain fru expression in ORNs. Such a dual mechanism of fru regulation in ORNs might be a trait of neurons driving plastic aspects of sex-specific behaviors

<i>fru</i> expression in adult Or47b ORNs requires Or47b function.

<p><b>(A)</b> Heterozygous <i>Or47b</i> mutant antennae (3–5 d old) expressing <i>fruGal4 40XUASCD8GFP</i> <b>(A)</b> and <i>OR47b-CD2</i> <b>(A’)</b>. <b>(A”)</b> shows the merge of two images. <b>(B)</b> Homozygous <i>Or47b</i> mutant antennae (3–5 d old). <b>(C)</b> Overexpression of <i>UAS-Or47b</i> under the control of <i>fruGal4</i> in <i>Or47b</i> mutants (14 d old). <b>(D)</b> Overexpression of <i>UAS-Or88a</i> under the control of <i>fruGal4</i> in <i>Or47b</i> mutants (14 d old).</p> <p>GENOTYPES:</p> <p>A–A”: <i>Or47b-CD2 Or47b</i>^<i>2</i><i>/+;fru</i>^<i>GAL4</i> <i>UAS-40XCD8GFP/+</i></p> <p>B–B”: <i>Or47b-CD2 Or47b</i>^<i>2</i><i>/or47b</i>^<i>2</i>;<i>fru</i>^<i>GAL4</i> <i>UAS-40XCD8GFP/+</i></p> <p>C: <i>Or47b</i>^<i>2</i><i>/Or47b</i>^<i>2</i>;<i>fru</i>^<i>GAL4</i> <i>UAS-40XCD8GFP/UAS-Or47b</i></p> <p>D: <i>Or47b-CD2 Or47b</i>^<i>2</i><i>/Or47b</i>^<i>2</i>;<i>fru</i>^<i>GAL4</i> <i>UAS-40XCD8GFP/UAS-Or88a</i></p

qPCR primers.

<p>qPCR primers.</p

<i>fru</i>-positive OR expression in at4 sensilla expands to developmentally related <i>fru</i>-negative ORNs in <i>alh</i> mutants.

<p><b> A)</b> Adult antennae and brains labeled with <i>Or47bGal4 UAS-CD8GFP</i> (green) in wild type and <i>alh</i> mutant clones. Magenta staining in brains is against N-cadherin, a neuropil marker. <b>B)</b> Adult antennae and brains labeled with <i>Or88aGal4 UAS-CD8GFP</i> in wild-type and <i>alh</i> mutant clones. <b>C)</b> Adult antennae and brains labeled with <i>Or65aGal4 UAS-CD8GFP</i> in wild-type and <i>alh</i> mutant clones. <b>D–G)</b> Quantification of cell bodies observed in the adult antennae of WT and <i>alh</i> mutant clones. For all graphs, asterisks indicate significant (<i>p</i> < .05) differences from wild type. Error bars represent standard error of the mean (SEM). An ANOVA was performed for each cell type and followed with Tukey’s Honest Significant Difference (HSD)—see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002443#sec015" target="_blank">Materials and Methods</a>. <b>D)</b> Total <i>Or47b</i>-positive cells. Wild type flies were significantly different from both <i>alh</i> conditions (<i>p</i> < .0001). <i>n</i> = 10–40. All count data may be found in the Supporting Information as <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002443#pbio.1002443.s001" target="_blank">S1 Data</a>. <b>E)</b> Total <i>Or88a</i>-positive cells. Both <i>alh</i> conditions were significantly different from wild-type males (<i>p</i> < .0001). <i>n</i> = 27 − 57. <b>F)</b> Total <i>Or65a</i>-positive cells. Both <i>alh</i> conditions were significantly different from wild-type males (<i>p</i> < .05). <i>n</i> = 9–27. <b>G)</b> Total <i>or47b</i>-positive clusters, normalized by total <i>Or47b</i>-positive cells. Wild type flies were significantly different from all <i>alh</i> conditions (<i>p</i> < .0001). <i>n</i> = 10–40. <b>H)</b> Model: in <i>alh</i> mutants, the <i>Or47b</i> odorant receptor expression is expanded to the other ORNs in the at4 sensilla, at the expense of their native OR expression, but the axons of these ORNs continue to target their original locations in the antennal lobe.</p> <p>GENOTYPES:</p> <p>A) <i>eyflp</i>; <i>Or47bGal4/UAS-CD8GFP</i>; <i>FRT82/FRT82Gal80E2F</i>,</p> <p><i>eyflp</i>; <i>Or47bGal4</i>/<i>UAS-CD8GFP</i>; <i>FRT82alh</i>^<i>1353</i>/<i>FRT82Gal80E2F</i>,</p> <p><i>eyflp</i>; <i>Or47bGal4</i>/<i>UAS-CD8GFP</i>; <i>FRT82alh</i>^<i>j8c8</i>/<i>FRT82Gal80E2F</i></p> <p>B) <i>eyflp</i>; <i>Or88aGal4/UAS-CD8GFP</i>; <i>FRT82/FRT82Gal80E2F</i>,</p> <p><i>eyflp</i>; <i>Or88aGal4</i>/<i>UAS-CD8GFP</i>; <i>FRT82alh</i>^<i>1353</i>/<i>FRT82Gal80E2F</i>,</p> <p><i>eyflp</i>; <i>Or88aGal4</i>/<i>UAS-CD8GFP</i>; <i>FRT82alh</i>^<i>j8c8</i>/<i>FRT82Gal80E2F</i></p> <p>C) <i>eyflp</i>; <i>Or65aGal4/UAS-CD8GFP</i>; <i>FRT82/FRT82Gal80E2F</i>,</p> <p><i>eyflp</i>; <i>Or65aGal4</i>/<i>UAS-CD8GFP</i>; <i>FRT82alh</i>^<i>1353</i>/<i>FRT82Gal80E2F</i>,</p> <p><i>eyflp</i>; <i>Or65aGal4</i>/<i>UAS-CD8GFP</i>; <i>FRT82alh</i>^<i>j8c8</i>/<i>FRT82Gal80E2F</i></p