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141 Publications

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    03/01/12 | Preparation and mounting of adult Drosophila structures in Canada balsam.
    Stern DL, Sucena E
    Cold Spring Harb Protoc. 2012 Mar;2012(3):373-5. doi: 10.1101/pdb.prot067389

    The Drosophila cuticle carries a rich array of morphological details. Thus, cuticle examination has had a central role in the history of genetics. To prepare fine "museum-quality," permanent slides, it is best to mount specimens in Canada Balsam. It is difficult to give precise recipes for Canada Balsam, because every user seems to prefer a slightly different viscosity. Dilute solutions spread easily and do not dry too rapidly while mounting specimens. The disadvantage is that there is actually less Balsam in a "drop" of the solution, and when dried, it can contract from the sides of the coverslip, sometimes disturbing the specimen. Unfortunately, there is no substitute for experience when using Canada Balsam. This protocol describes a procedure for mounting adult cuticles in Canada Balsam.

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    04/01/12 | Investigation of the role of Aubergine RNA-binding proteins in the reproductive plasticity of the pea aphid, Acyrthosiphon pisum
    A Abdelhady , R Cortes , S Musumeci , D Srinivasan , S Shigenobu , D Stern , S Kobayashi
    Society for Integrative and Comparative Biology. 01/2012;52:E202-E202

    Environmental changes can elicit alterations in the form, behavior and/or physiology of all species, and this developmental response to environment is known as phenotypic plasticity. Despite its ubiquity, the molecular basis for phenotypic plasticity is not fully understood. The pea aphid, Acyrthosiphon pisum, serves as a model for an extreme form of phenotypic plasticity, known as polyphenism. Changes in photoperiod stimulate a switch in female aphid reproductive mode from asexual to sexual reproduction over the course of one generation without changes in genotype. This reproductive polyphenism results in female aphids with ovaries of one of two types: sexual ovaries (producing haploid oocytes via meiosis), or asexual ovaries (producing identical diploid aphid clones via parthenogenesis). To better understand how aphid ovaries could produce different outputs, we surveyed the transcriptomes of sexual and asexual ovaries using RNA-seq. Among genes that exhibited greater than two-fold differences in gene expression between sexual and asexual ovaries, we identified several aubergine paralogs, which encode for germline-specific members of the Argonaute small RNA-binding protein family. The A. pisum genome contains eight aubergine paralogs and at least two piwi paralogs. We are currently comparing the expression patterns of these aphid aubergine paralogs between asexual and sexual aphid ovaries. Aubergine proteins in other species are thought to help suppress the activity of transposable elements, which are found in high quantities throughout the A. pisum genome. Together, these experiments will help elucidate a potential relationship between aubergine paralogs and aphid reproductive plasticity.

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    01/01/12 | Evolution of multiple additive loci caused divergence between Drosophila yakuba and D. santomea in wing rowing during male courtship.
    Cande J, Andolfatto P, Prud'homme B, Stern DL, Gompel N
    PLoS One. 2012;7(8):e43888. doi: 10.1371/journal.pone.0043888

    In Drosophila, male flies perform innate, stereotyped courtship behavior. This innate behavior evolves rapidly between fly species, and is likely to have contributed to reproductive isolation and species divergence. We currently understand little about the neurobiological and genetic mechanisms that contributed to the evolution of courtship behavior. Here we describe a novel behavioral difference between the two closely related species D. yakuba and D. santomea: the frequency of wing rowing during courtship. During courtship, D. santomea males repeatedly rotate their wing blades to face forward and then back (rowing), while D. yakuba males rarely row their wings. We found little intraspecific variation in the frequency of wing rowing for both species. We exploited multiplexed shotgun genotyping (MSG) to genotype two backcross populations with a single lane of Illumina sequencing. We performed quantitative trait locus (QTL) mapping using the ancestry information estimated by MSG and found that the species difference in wing rowing mapped to four or five genetically separable regions. We found no evidence that these loci display epistasis. The identified loci all act in the same direction and can account for most of the species difference.

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    01/01/12 | Rapid mounting of adult Drosophila structures in Hoyer's medium.
    Stern DL, Sucena E
    Cold Spring Harb Protoc. 2012 Jan;2012(1):107-9. doi: 10.1101/pdb.prot067371

    The Drosophila cuticle carries a rich array of morphological details. Thus, cuticle examination has had a central role in the history of genetics. This protocol describes a procedure for mounting adult cuticles in Hoyer's medium, a useful mountant for both larval and adult cuticles. The medium digests soft tissues rapidly, leaving the cuticle cleared for observation. In addition, samples can be transferred directly from water to Hoyer's medium. However, specimens mounted in Hoyer's medium degrade over time. For example, the fine denticles on the larval dorsum are best observed soon after mounting; they begin to fade after 1 week, and can disappear completely after several months. More robust features, such as the ventral denticle belts, will persist for a longer period of time. Because adults cannot profitably be mounted whole in Hoyer's medium, some dissection is necessary.

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    11/01/11 | Preparation of cuticles from feeding Drosophila larvae.
    Stern DL, Sucena E
    Cold Spring Harb Protoc. 2011 Nov;2011(11):1394-8. doi: 10.1101/pdb.prot066498

    The Drosophila cuticle carries a rich array of morphological details. Thus, cuticle examination has had a central role in the history of genetics. Studies of the Drosophila cuticle have focused mainly on first-instar larvae and adult cuticular morphology. Although the cuticles of second- and third-instar larvae are strikingly different from those of the first instar, these differences have been poorly studied. This protocol describes three methods for preparing cuticles from fed larvae. One commonly used procedure involves manually pricking the larvae. A simpler method for preparing larval cuticles is to burst the larvae once they have been mounted. This method is used for first- and second-instar larvae and does not require pricking; it removes the gut contents by "popping" the rear of the embryo using pressure from the coverslip. If just the right amount of medium is used, the coverslip will be pulled toward the slide, applying pressure on the samples. The larvae usually burst from their posterior ends. Also presented is an alternative procedure designed specifically for the use with third-instar larvae, although the "pricking" method can be used at this stage.

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    09/01/11 | Preparation of cuticles from unhatched first-instar Drosophila larvae.
    Stern DL, Sucena E
    Cold Spring Harb Protoc. 2011 Sep;2011(9):. doi: 10.1101/pdb.prot065532

    The finely sculpted cuticle of Drosophila carries a rich array of morphological details. Thus, cuticle examination has had a central role in the history of genetics. Studies of the Drosophila cuticle have focused mainly on first-instar larvae and adult cuticular morphology. This protocol describes the preparation of cuticles from larvae that have not yet hatched from the egg. It is designed for sampling all eggs laid by one or more females. This can be particularly useful, for example, when a mutation produces embryos that are unable to hatch from the egg.

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    06/30/11 | Morphological evolution caused by many subtle-effect substitutions in regulatory DNA.
    Frankel N, Erezyilmaz DF, McGregor AP, Wang S, Payre Fc, Stern DL
    Nature. 2011 Jun 30;474(7353):598-603. doi: 10.1038/nature10200

    Morphology evolves often through changes in developmental genes, but the causal mutations, and their effects, remain largely unknown. The evolution of naked cuticle on larvae of Drosophila sechellia resulted from changes in five transcriptional enhancers of shavenbaby (svb), a transcript of the ovo locus that encodes a transcription factor that governs morphogenesis of microtrichiae, hereafter called ’trichomes’. Here we show that the function of one of these enhancers evolved through multiple single-nucleotide substitutions that altered both the timing and level of svb expression. The consequences of these nucleotide substitutions on larval morphology were quantified with a novel functional assay. We found that each substitution had a relatively small phenotypic effect, and that many nucleotide changes account for this large morphological difference. In addition, we observed that the substitutions had non-additive effects. These data provide unprecedented resolution of the phenotypic effects of substitutions and show how individual nucleotide changes in a transcriptional enhancer have caused morphological evolution.

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    04/01/11 | Multiplexed shotgun genotyping for rapid and efficient genetic mapping.
    Andolfatto P, Davison D, Erezyilmaz D, Hu TT, Mast J, Sunayama-Morita T, Stern DL
    Genome Research. 2011 Apr;21(4):610-7. doi: 10.1101/gr.115402.110

    We present a new approach to genotyping based on multiplexed shotgun sequencing that can identify recombination breakpoints in a large number of individuals simultaneously at a resolution sufficient for most mapping purposes, such as quantitative trait locus (QTL) mapping and mapping of induced mutations. We first describe a simple library construction protocol that uses just 10 ng of genomic DNA per individual and makes the approach accessible to any laboratory with standard molecular biology equipment. Sequencing this library results in a large number of sequence reads widely distributed across the genomes of multiplexed bar-coded individuals. We develop a Hidden Markov Model to estimate ancestry at all genomic locations in all individuals using these data. We demonstrate the utility of the approach by mapping a dominant marker allele in D. simulans to within 105 kb of its true position using 96 F1-backcross individuals genotyped in a single lane on an Illumina Genome Analyzer. We further demonstrate the utility of our method by genetically mapping more than 400 previously unassembled D. simulans contigs to linkage groups and by evaluating the quality of targeted introgression lines. At this level of multiplexing and divergence between strains, our method allows estimation of recombination breakpoints to a median of 38-kb intervals. Our analysis suggests that higher levels of multiplexing and/or use of strains with lower levels of divergence are practicable.

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    08/01/10 | Dynamics of genome evolution in facultative symbionts of aphids.
    Degnan PH, Leonardo TE, Cass BN, Hurwitz B, Stern D, Gibbs RA, Richards S, Moran NA
    Environmental Microbiology. 2010 Aug;12(8):2060-9. doi: 10.1111/j.1462-2920.2009.02085.x

    Aphids are sap-feeding insects that host a range of bacterial endosymbionts including the obligate, nutritional mutualist Buchnera plus several bacteria that are not required for host survival. Among the latter, ’Candidatus Regiella insecticola’ and ’Candidatus Hamiltonella defensa’ are found in pea aphids and other hosts and have been shown to protect aphids from natural enemies. We have sequenced almost the entire genome of R. insecticola (2.07 Mbp) and compared it with the recently published genome of H. defensa (2.11 Mbp). Despite being sister species the two genomes are highly rearranged and the genomes only have \~{}55% of genes in common. The functions encoded by the shared genes imply that the bacteria have similar metabolic capabilities, including only two essential amino acid biosynthetic pathways and active uptake mechanisms for the remaining eight, and similar capacities for host cell toxicity and invasion (type 3 secretion systems and RTX toxins). These observations, combined with high sequence divergence of orthologues, strongly suggest an ancient divergence after establishment of a symbiotic lifestyle. The divergence in gene sets and in genome architecture implies a history of rampant recombination and gene inactivation and the ongoing integration of mobile DNA (insertion sequence elements, prophage and plasmids).

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    07/22/10 | Phenotypic robustness conferred by apparently redundant transcriptional enhancers.
    Frankel N, Davis GK, Vargas D, Wang S, Payre F, Stern DL
    Nature. 2010 Jul 22;466(7305):490-3. doi: 10.1038/nature09158

    Genes include cis-regulatory regions that contain transcriptional enhancers. Recent reports have shown that developmental genes often possess multiple discrete enhancer modules that drive transcription in similar spatio-temporal patterns: primary enhancers located near the basal promoter and secondary, or ’shadow’, enhancers located at more remote positions. It has been proposed that the seemingly redundant activity of primary and secondary enhancers contributes to phenotypic robustness. We tested this hypothesis by generating a deficiency that removes two newly discovered enhancers of shavenbaby (svb, a transcript of the ovo locus), a gene encoding a transcription factor that directs development of Drosophila larval trichomes. At optimal temperatures for embryonic development, this deficiency causes minor defects in trichome patterning. In embryos that develop at both low and high extreme temperatures, however, absence of these secondary enhancers leads to extensive loss of trichomes. These temperature-dependent defects can be rescued by a transgene carrying a secondary enhancer driving transcription of the svb cDNA. Finally, removal of one copy of wingless, a gene required for normal trichome patterning, causes a similar loss of trichomes only in flies lacking the secondary enhancers. These results support the hypothesis that secondary enhancers contribute to phenotypic robustness in the face of environmental and genetic variability.

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