Dynamic Interpretation of Hedgehog Signaling in the Drosophila Wing Disc

Morphogens are classically defined as molecules that control patterning by acting at a distance to regulate gene expression in a concentration-dependent manner. In the Drosophila wing imaginal disc, secreted Hedgehog (Hh) forms an extracellular gradient that organizes patterning along the anterior–posterior axis and specifies at least three different domains of gene expression. Although the prevailing view is that Hh functions in the Drosophila wing disc as a classical morphogen, a direct correspondence between the borders of these patterns and Hh concentration thresholds has not been demonstrated. Here, we provide evidence that the interpretation of Hh signaling depends on the history of exposure to Hh and propose that a single concentration threshold is sufficient to support multiple outputs. Using mathematical modeling, we predict that at steady state, only two domains can be defined in response to Hh, suggesting that the boundaries of two or more gene expression patterns cannot be specified by a static Hh gradient. Computer simulations suggest that a spatial “overshoot” of the Hh gradient occurs, i.e., a transient state in which the Hh profile is expanded compared to the Hh steady-state gradient. Through a temporal examination of Hh target gene expression, we observe that the patterns initially expand anteriorly and then refine, providing in vivo evidence for the overshoot. The Hh gene network architecture suggests this overshoot results from the Hh-dependent up-regulation of the receptor, Patched (Ptc). In fact, when the network structure was altered such that the ptc gene is no longer up-regulated in response to Hh-signaling activation, we found that the patterns of gene expression, which have distinct borders in wild-type discs, now overlap. Our results support a model in which Hh gradient dynamics, resulting from Ptc up-regulation, play an instructional role in the establishment of patterns of gene expression

Altered urothelial ATP signaling in a major subset of human overactive bladder patients with pyuria

Overactive Bladder (OAB) is an idiopathic condition, characterized by urgency, urinary frequency, and urgency incontinence, in the absence of routinely traceable urinary infection. We have described microscopic pyuria (?10 wbc/?l) in patients suffering from the worst symptoms. It is established that inflammation is associated with increased ATP release from epithelial cells, and extracellular ATP originating from the urothelium following increased hydrostatic pressure is a mediator of bladder sensation. Here, using bladder biopsy samples, we have investigated urothelial ATP signaling in OAB patients with microscopic pyuria. Basal, but not stretch-evoked, release of ATP was significantly greater from the urothelium of OAB patients with pyuria than from non-OAB patients or OAB patients without pyuria (<10 wbc/?l). Basal ATP release from the urothelium of OAB patients with pyuria was inhibited by the P2 receptor antagonist suramin and abolished by the hemichannel blocker carbenoxolone, which differed from stretch-activated ATP release. Altered P2 receptor expression was evident in the urothelium from pyuric OAB patients. Furthermore, intracellular bacteria were visualized in shed urothelial cells from ?80% of OAB patients with pyuria. These data suggest that increased ATP release from the urothelium, involving bacterial colonization, may play a role in the heightened symptoms associated with pyuric OAB patients

Towards a dated molecular phylogeny of the Tanypodinae (Chironomidae, Diptera)

A dated molecular phylogeny is proposed for the Tanypodinae, a diverse subfamily of Chironomidae (Diptera). We used molecular data from fragments of one ribosomal gene (28S), one nuclear protein-coding gene (CAD), and one mitochondrial protein-coding gene (COI), analysed using mixed model Bayesian and maximum likelihood inference methods. All proposed tribes were sampled, namely, Anatopyniini, Clinotanypodini, Coelopyniini, Fittkauimyiini, Macropelopiini, Natarsiini, Pentaneurini, Procladiini and Tanypodini. A multilocus dataset of 1938 characters was compiled from 123 individuals including outgroups. Monophyly was supported for all tribes although some relationships were not robust. Relationships between tribes and some genus groups are highly congruent with a morphology-based estimate. Relationships within tribe Pentaneurini mostly find weak support, yet previously hypothesised groupings and monophyly or lack thereof in well-sampled genera are revealed. The tempo of diversification of the family was deduced by divergence time analysis (BEAST). Origination of a subfamily stem group in the late Jurassic to early Cretaceous was inferred, with all tribes and many genera of Pentaneurini originating and diversifying in the Cretaceous. Some nodes are biogeographically informative. Gene sections supported the backbone, but more extensive sampling is needed to estimate shallower phylogenies and to better understand the tempo and diversification of Tanypodinae

A transcriptome-based analytical workflow for identifying loci for species diagnosis: A case study with Bactrocera fruit flies (Insecta: Tephritidae)

Development of novel molecular methods for accurate and economical identification of species has become critical both for pure biological research and a wide range of applied areas. The most widely used current molecular diagnostic tool, the mitochondrial cytochrome c oxidase subunit 1 gene (COI), the so-called ‘DNA barcode’, has been highly criticised and is known to be ineffective at distinguishing species in many groups. Alternative markers are needed to circumvent these issues and provide diagnosticians with a greater range of tools for making accurate identifications. To address this, we describe here a novel analytical workflow for diagnostic marker development that utilises near-genomic scale data to search for potential informative loci. The workflow takes advantage of orthologous gene databases, in combination with tests of phylogenetic resolution, and benchmarking of nucleotide variation against COI, to determine putative loci that might outperform COI. We use transcriptomes of 14 tephritid fruit flies, and especially the taxonomically complex genus Bactrocera, as a case study. Of 1646 orthologs searched, our workflow retained a total of five loci following our conservative filtering strategy. One locus, POP4, had strong potential as a novel diagnostic marker for Bactrocera fruit flies. POP4 discriminates most species in the training set of taxa, but like COI fails to separate the sibling species B. tryoni and B. neohumeralis. Further validation of this potential new marker against a broader taxonomic sample is ongoing. We advocate that this simple and efficient workflow is, with minor modification, customisable for diagnostic development in almost any taxonomic group

A comprehensive phylogeny helps clarify the evolutionary history of host breadth and lure response in the Australian Dacini fruit flies (Diptera: Tephritidae)

The tribe Dacini (Diptera: Tephritidae) contains over 930 recognised species and has been widely studied due to the economic importance of some taxa, such as the Oriental fruit fly Bactrocera dorsalis. Despite the attention this group has received, very few phylogenetic reconstructions have comprehensively sampled taxa from a single biogeographic region, thereby limiting our capacity to address more targeted evolutionary questions. To study the evolution of diet breadth and male lure response, two key traits fundamental to understanding dacine diversity and the biology of pest taxa, we analysed 273 individuals representing 144 described species from Australia (80% continental coverage), the Pacific, and select close relatives from South-east Asia to estimate a dated molecular phylogenetic reconstruction of the Dacini. We utilised seven loci with a combined total of 4,332 nucleotides, to estimate both Bayesian and Maximum Likelihood phylogenies of the tribe. Consistent with other molecular phylogenies of the tribe, there was a high level of disagreement between the placement of species in the phylogeny and their current subgeneric and species-complex level taxonomies. The Australian fauna exhibit high levels of endemism, with radiations of both exclusively Australian clades, and clades that originate elsewhere (e.g. the Bactrocera dorsalis species group). Bidirectional movement of species has occurred between Papua New Guinea and Australia, with evidence for multiple incursions over evolutionary time. The Bactrocera aglaiae species group emerged sister to all other Bactrocera species examined. Divergence time estimates were ∼ 30 my younger than previously reported for this group, with the tribe diverging from its most recent common ancestor ∼ 43 mya. Ancestral trait reconstruction and tests for trait phylogenetic signal revealed a strong signal for the evolution of male lure response across the tree, with cue-lure/raspberry ketone lure response the ancestral trait. Methyl eugenol response has arisen on multiple, independent occasions. The evolution of host breadth exhibited a weaker signal; yet, basal groups were more likely to be host specialists. Both the evolution of lure response and host fruit use provide predictive information for the outbreak management of understudied pest fruit flies for which direct inference of these features may be lacking. Our results, which parallel those of earlier research into the closely-related African Dacus spp., demonstrate how geographically focussed taxon coverage allows Dacini phylogenetics to more explicitly test evolutionary hypotheses, thereby progressing our understanding of the evolution of this highly diverse and recently-radiated group of flies

Flow cytometry analysis of P(3HB) production in <i>phaCAB</i>-engineered <i>E</i>. <i>coli</i> from waste-media cultures.

<p><i>E</i>. <i>coli</i> MG1655 transformed with either empty vector [EV], native [N], constitutive [C] or hybrid [H] <i>phaCAB</i> constructs were cultured in 5 ml of waste-media for 36 h at 37°C. P(3HB) content was assessed via flow cytometry analysis of Nile Red staining. (<b>A</b>) Representative forward scatter (FSC) and side scatter (SSC) contour plots. (<b>B</b>) Representative histogram (FL-5). (<b>C</b>) Normalized fluorescence of Nile Red stained <i>phaCAB</i>-engineered <i>E</i>. <i>coli</i>, from three independent experiments. Error bars, +/- the standard deviation. Student t-test, *P<0.05 and ***P <0.001.</p

Simulated P(3HB) production in <i>phaCAB</i>-engineered <i>E</i>. <i>coli</i>.

<p>In order to simulate P(3HB) production in <i>phaCAB</i>-engineered <i>E</i>. <i>coli</i>, a P(3HB) synthesis model was constructed using the Simbiology toolbox of Matlab. Using this model the flux of several metabolites and species were simulated in order to identify aspects of the system that could be selectively tuned to increase the production of P(3HB). From these analyses, several novel <i>phaCAB</i> operons were designed. These data show the simulated P(3HB) production across several different <i>phaCAB</i> operon designs, where <i>phaCAB</i> expression is under the control of the indicated Anderson constitutive promoters.</p