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67 Publications

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    Cardona Lab
    09/01/05 | An in situ hybridization protocol for planarian embryos: monitoring myosin heavy chain gene expression.
    Cardona A, Fernández J, Solana J, Romero R
    Development Genes & Evolution. 2005 Sep;215(9):482-88. doi: 10.1007/s00427-005-0003-1

    The monitoring of gene expression is fundamental for understanding developmental biology. Here we report a successful experimental protocol for in situ hybridization in both whole-mount and sectioned planarian embryos. Conventional in situ hybridization techniques in developmental biology are used on whole-mount preparations. However, given that the inherent lack of external morphological markers in planarian embryos hinders the proper interpretation of gene expression data in whole-mount preparations, here we used sectioned material. We discuss the advantages of sectioned versus whole-mount preparations, namely, better probe penetration, improved tissue preservation, and the possibility to interpret gene expression in relation to internal morphological markers such as the epidermis, the embryonic and definitive pharynges, and the gastrodermis. Optimal fixatives and embedding methods for sectioning are also discussed.

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    09/01/05 | Molecular genetic analysis of the yeast repressor Rfx1/Crt1 reveals a novel two-step regulatory mechanism.
    Zhang Z, Reese JC
    Molecular and Cellular Biology. 2005 Sep;25(17):7399-411. doi: 10.1128/MCB.25.17.7399-7411.2005

    In Saccharomyces cerevisiae, the repressor Crt1 and the global corepressor Ssn6-Tup1 repress the DNA damage-inducible ribonucleotide reductase (RNR) genes. Initiation of DNA damage signals causes the release of Crt1 and Ssn6-Tup1 from the promoter, coactivator recruitment, and derepression of transcription, indicating that Crt1 plays a crucial role in the switch between gene repression and activation. Here we have mapped the functional domains of Crt1 and identified two independent repression domains and a region required for gene activation. The N terminus of Crt1 is the major repression domain, it directly binds to the Ssn6-Tup1 complex, and its repression activities are dependent upon Ssn6-Tup1 and histone deacetylases (HDACs). In addition, we identified a C-terminal repression domain, which is independent of Ssn6-Tup1 and HDACs and functions at native genes in vivo. Furthermore, we show that TFIID and SWI/SNF bind to a region within the N terminus of Crt1, overlapping with but distinct from the Ssn6-Tup1 binding and repression domain, suggesting that Crt1 may have activator functions. Crt1 mutants were constructed to dissect its activator and repressor functions. All of the mutants were competent for repression of the DNA damage-inducible genes, but a majority were "derepression-defective" mutants. Further characterization of these mutants indicated that they are capable of receiving DNA damage signals and releasing the Ssn6-Tup1 complex from the promoter but are selectively impaired for TFIID and SWI/SNF recruitment. These results imply a two-step activation model of the DNA damage-inducible genes and that Crt1 functions as a signal-dependent dual-transcription activator and repressor that acts in a transient manner.

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    09/01/05 | The temporal requirements for insulin signaling during development in Drosophila.
    Shingleton AW, Das J, Vinicius L, Stern DL
    PLoS Biol. 2005 Sep;3(9):e289. doi: 10.1371/journal.pbio.0030289

    Recent studies have indicated that the insulin-signaling pathway controls body and organ size in Drosophila, and most metazoans, by signaling nutritional conditions to the growing organs. The temporal requirements for insulin signaling during development are, however, unknown. Using a temperature-sensitive insulin receptor (Inr) mutation in Drosophila, we show that the developmental requirements for Inr activity are organ specific and vary in time. Early in development, before larvae reach the "critical size" (the size at which they commit to metamorphosis and can complete development without further feeding), Inr activity influences total development time but not final body and organ size. After critical size, Inr activity no longer affects total development time but does influence final body and organ size. Final body size is affected by Inr activity from critical size until pupariation, whereas final organ size is sensitive to Inr activity from critical size until early pupal development. In addition, different organs show different sensitivities to changes in Inr activity for different periods of development, implicating the insulin pathway in the control of organ allometry. The reduction in Inr activity is accompanied by a two-fold increase in free-sugar levels, similar to the effect of reduced insulin signaling in mammals. Finally, we find that varying the magnitude of Inr activity has different effects on cell size and cell number in the fly wing, providing a potential linkage between the mode of action of insulin signaling and the distinct downstream controls of cell size and number. We present a model that incorporates the effects of the insulin-signaling pathway into the Drosophila life cycle. We hypothesize that the insulin-signaling pathway controls such diverse effects as total developmental time, total body size and organ size through its effects on the rate of cell growth, and proliferation in different organs.

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    08/11/05 | The hangover gene defines a stress pathway required for ethanol tolerance development.
    Scholz H, Franz M, Heberlein U
    Nature. 2005 Aug 11;436(7052):845-7. doi: 10.1038/nature03864

    Repeated alcohol consumption leads to the development of tolerance, simply defined as an acquired resistance to the physiological and behavioural effects of the drug. This tolerance allows increased alcohol consumption, which over time leads to physical dependence and possibly addiction. Previous studies have shown that Drosophila develop ethanol tolerance, with kinetics of acquisition and dissipation that mimic those seen in mammals. This tolerance requires the catecholamine octopamine, the functional analogue of mammalian noradrenaline. Here we describe a new gene, hangover, which is required for normal development of ethanol tolerance. hangover flies are also defective in responses to environmental stressors, such as heat and the free-radical-generating agent paraquat. Using genetic epistasis tests, we show that ethanol tolerance in Drosophila relies on two distinct molecular pathways: a cellular stress pathway defined by hangover, and a parallel pathway requiring octopamine. hangover encodes a large nuclear zinc-finger protein, suggesting a role in nucleic acid binding. There is growing recognition that stress, at both the cellular and systemic levels, contributes to drug- and addiction-related behaviours in mammals. Our studies suggest that this role may be conserved across evolution.

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    08/01/05 | A major role for zygotic hunchback in patterning the Nasonia embryo.
    Pultz MA, Westendorf L, Gale SD, Hawkins K, Lynch J, Pitt JN, Reeves NL, Yao JC, Small S, Desplan C, Leaf DS
    Development . 2005 Aug;132(16):3705-15. doi: 10.1242/dev.01939

    Developmental genetic analysis has shown that embryos of the parasitoid wasp Nasonia vitripennis depend more on zygotic gene products to direct axial patterning than do Drosophila embryos. In Drosophila, anterior axial patterning is largely established by bicoid, a rapidly evolving maternal-effect gene, working with hunchback, which is expressed both maternally and zygotically. Here, we focus on a comparative analysis of Nasonia hunchback function and expression. We find that a lesion in Nasonia hunchback is responsible for the severe zygotic headless mutant phenotype, in which most head structures and the thorax are deleted, as are the three most posterior abdominal segments. This defines a major role for zygotic Nasonia hunchback in anterior patterning, more extensive than the functions described for hunchback in Drosophila or Tribolium. Despite the major zygotic role of Nasonia hunchback, we find that it is strongly expressed maternally, as well as zygotically. Nasonia Hunchback embryonic expression appears to be generally conserved; however, the mRNA expression differs from that of Drosophila hunchback in the early blastoderm. We also find that the maternal hunchback message decays at an earlier developmental stage in Nasonia than in Drosophila, which could reduce the relative influence of maternal products in Nasonia embryos. Finally, we extend the comparisons of Nasonia and Drosophila hunchback mutant phenotypes, and propose that the more severe Nasonia hunchback mutant phenotype may be a consequence of differences in functionally overlapping regulatory circuitry.

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    08/01/05 | Cellular mechanisms of dendrite pruning in Drosophila: insights from in vivo time-lapse of remodeling dendritic arborizing sensory neurons.
    Williams DW, Truman JW
    Development. 2005 Aug;132(16):3631-42. doi: 10.1242/dev.01928

    Regressive events that refine exuberant or inaccurate connections are critical in neuronal development. We used multi-photon, time-lapse imaging to examine how dendrites of Drosophila dendritic arborizing (da) sensory neurons are eliminated during early metamorphosis, and how intrinsic and extrinsic cellular mechanisms control this deconstruction. Removal of the larval dendritic arbor involves two mechanisms: local degeneration and branch retraction. In local degeneration, major branch severing events entail focal disruption of the microtubule cytoskeleton, followed by thinning of the disrupted region, severing and fragmentation. Retraction was observed at distal tips of branches and in proximal stumps after severing events. The pruning program of da neuron dendrites is steroid induced; cell-autonomous dominant-negative inhibition of steroid action blocks local degeneration, although retraction events still occur. Our data suggest that steroid-induced changes in the epidermis may contribute to dendritic retraction. Finally, we find that phagocytic blood cells not only engulf neuronal debris but also attack and sever intact branches that show signs of destabilization.

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    08/01/05 | Feature selection based on mutual information: criteria of max-dependency, max-relevance, and min-redundancy.
    Peng H, Long F, Ding C
    IEEE Transactions on Pattern Analysis and Machine Intelligence. 2005 Aug;27(8):1226-38. doi: 10.1007/s12021-010-9090-x

    Feature selection is an important problem for pattern classification systems. We study how to select good features according to the maximal statistical dependency criterion based on mutual information. Because of the difficulty in directly implementing the maximal dependency condition, we first derive an equivalent form, called minimal-redundancy-maximal-relevance criterion (mRMR), for first-order incremental feature selection. Then, we present a two-stage feature selection algorithm by combining mRMR and other more sophisticated feature selectors (e.g., wrappers). This allows us to select a compact set of superior features at very low cost. We perform extensive experimental comparison of our algorithm and other methods using three different classifiers (naive Bayes, support vector machine, and linear discriminate analysis) and four different data sets (handwritten digits, arrhythmia, NCI cancer cell lines, and lymphoma tissues). The results confirm that mRMR leads to promising improvement on feature selection and classification accuracy.

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    Baker Lab
    07/21/05 | Male-specific fruitless specifies the neural substrates of Drosophila courtship behaviour.
    Manoli DS, Foss M, Villella A, Taylor BJ, Hall JC, Baker BS
    Nature. 2005 Jul 21;436(7049):395-400. doi: 10.1038/nature03859

    Robust innate behaviours are attractive systems for genetically dissecting how environmental cues are perceived and integrated to generate complex behaviours. During courtship, Drosophila males engage in a series of innate, stereotyped behaviours that are coordinated by specific sensory cues. However, little is known about the specific neural substrates mediating this complex behavioural programme. Genetic, developmental and behavioural studies have shown that the fruitless (fru) gene encodes a set of male-specific transcription factors (FruM) that act to establish the potential for courtship in Drosophila. FruM proteins are expressed in approximately 2% of central nervous system neurons, at least one subset of which coordinates the component behaviours of courtship. Here we have inserted the yeast GAL4 gene into the fru locus by homologous recombination and show that (1) FruM is expressed in subsets of all peripheral sensory systems previously implicated in courtship, (2) inhibition of FruM function in olfactory system components reduces olfactory-dependent changes in courtship behaviour, (3) transient inactivation of all FruM-expressing neurons abolishes courtship behaviour, with no other gross changes in general behaviour, and (4) ’masculinization’ of FruM-expressing neurons in females is largely sufficient to confer male courtship behaviour. Together, these data demonstrate that FruM proteins specify the neural substrates of male courtship.

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    07/08/05 | Postsynaptic depolarization requirements for LTP and LTD: a critique of spike timing-dependent plasticity.
    Lisman J, Spruston N
    Nat Neurosci. 2005 Jul;8(7):839-41

    Long-term potentiation and long-term depression require postsynaptic depolarization, which many current models attribute to backpropagating action potentials. New experimental work suggests, however, that other mechanisms can lead to dendritic depolarization, and that backpropagating action potentials may be neither necessary nor sufficient for synaptic plasticity in vivo.

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    07/05/05 | Many-particle tracking with nanometer resolution in three dimensions by reflection interference contrast microscopy.
    Clack NG, Groves JT
    Langmuir: The ACS Journal of Surfaces and Colloids. 2005 Jul 5;21(14):6430-5. doi: 10.1021/la050372r

    We have developed and characterized a method, based on reflection interference contrast microscopy, to simultaneously determine the three-dimensional positions of multiple particles in a colloidal monolayer. To evaluate this method, the interaction of 6.8 microm (+/-5%) diameter lipid-derivatized silica microspheres with an underlying planar borosilicate substrate is studied. Measured colloidal height distributions are consistent with expectations for an electrostatically levitated colloidal monolayer. The precision of the method is analyzed using experimental techniques in addition to computational bootstrapping algorithms. In its present implementation, this technique achieves 16 nm lateral and 1 nm vertical precision.

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