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In JoVE (1)
Other Publications (4)
Articles by Diane Ramos in JoVE
In situ Protocol for Butterfly Pupal Wings Using Riboprobes
Diane Ramos1, Antonia Monteiro2
1Department of Biological Sciences, SUNY-University at Buffalo, 2Dept. Ecology and Evolutionary Biology, Yale University
In order to examine gene expression in the pupal wing tissue of Bicyclus anynana, we present an optimized protocol for in situ hybridizations using riboprobes. We also provide guidelines for the further optimization of this protocol for use in pupal wings of other Lepidopteran species.
Other articles by Diane Ramos on PubMed
Proceedings. Biological Sciences / The Royal Society. Aug, 2004 | Pubmed ID: 15503989
Ecological and evolutionary theory has frequently been inspired by the diversity of colour patterns on the wings of butterflies. More recently, these varied patterns have also become model systems for studying the evolution of developmental mechanisms. A technique that will facilitate our understanding of butterfly colour-pattern development is germline transformation. Germline transformation permits functional tests of candidate gene products and of cis-regulatory regions, and provides a means of generating new colour-pattern mutants by insertional mutagenesis. We report the successful transformation of the African satyrid butterfly Bicyclus anynana with two different transposable element vectors, Hermes and piggyBac, each carrying EGFP coding sequences driven by the 3XP3 synthetic enhancer that drives gene expression in the eyes. Candidate lines identified by screening for EGFP in adult eyes were later confirmed by PCR amplification of a fragment of the EGFP coding sequence from genomic DNA. Flanking DNA surrounding the insertions was amplified by inverse PCR and sequenced. Transformation rates were 5% for piggyBac and 10.2% for Hermes. Ultimately, the new data generated by these techniques may permit an integrated understanding of the developmental genetics of colour-pattern formation and of the ecological and evolutionary processes in which these patterns play a role.
BMC Developmental Biology. 2006 | Pubmed ID: 17090321
Butterfly and moth eyespots can share a similar appearance, involving multiple concentric rings of colored scales, but usually occurring in non-homologous positions on the wing. Within the butterflies, on the other hand, spots that share the same homologous position may not share the concentric ring structure; and, in butterfly species that have eyespots with concentric rings, ectopic eyespots with a similar ring structure can be induced by means of a simple epidermal wound. The extent to which all these eyespots, natural or induced, share similar genes and developmental mechanisms is investigated here by means of protein in-situ localizations in selected butterfly and moth species. In addition to looking at some of the transcription factors previously identified as being involved in eyespot formation, we also tested the involvement of candidate genes from the Wingless and TGF-beta signaling pathways as putative morphogens for eyespot development.
BMC Developmental Biology. 2006 | Pubmed ID: 17116248
Precise temporal and spatial regulation of transgene expression is a critical tool to investigate gene function in developing organisms. The most commonly used technique to achieve tight control of transgene expression, however, requires the use of specific DNA enhancers that are difficult to characterize in non-model organisms. Here, we sought to eliminate the need for this type of sequence-based gene regulation and to open the field of functional genetics to a broader range of organisms.
Molecular BioSystems. Aug, 2007 | Pubmed ID: 17639127
The extremely diverse lepidopteran wing patterns make useful models to study the evolution of development and the molecular changes that enable it. Until now, the implication of candidate genes in the differentiation of color patterns has relied primarily on correlational evidence, i.e., gene expression patterns in a developing wing mapping closely to the adult color pattern. The use of transgenic techniques in the Lepidoptera, including the manipulation of gene expression, will finally allow researchers to test hypotheses of gene function at various levels of the patterning hierarchy, from signaling ligands and transcription factors to pigment enzymes. Here we present an overview of transgenic techniques employed in lepidopteran systems and highlight areas where current and future research will provide exciting opportunities to deepen our understanding of the mechanisms of morphological evolution.