Wir danken Sean B. Carroll und Thomas Werner für die Bereitstellung von fliegen Bestände, Naoyuki Sicherung für Ausrüstung, Byung Seok Jin für seine Hilfe bei Dreharbeiten, Kiyokazu Agata für mentoring und Elizabeth Nakajima zur englischen Bearbeitung. Diese Arbeit wurde durch KAKENHI unterstützt 17K 19427 und Takeda Science Foundation.

<ol>
	<li>Carson, H. L., Hardy, D. E., Spieth, H. T., Stone, W. S., Hecht, M. K., Steere, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolutionary+biology+of+the+Hawaiian+Drosophilidae.">The evolutionary biology of the Hawaiian Drosophilidae.</a> Essays in evolution and genetics in honor of Theodosius Dobzhansky. , 437-543 (1970).</li><li>Markow, T. A., O'Grady, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: a guide to species identification and use. , (2006).</li><li>Patterson, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Drosophilidae of the southwest. 4313, 7-216 (1943).</li><li>Setoguchi, S., Takamori, H., Aotsuka, T., Sese, J., Ishikawa, Y., Matsuo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+dimorphism+and+courtship+behavior+in+Drosophila+prolongata.">Sexual dimorphism and courtship behavior in Drosophila prolongata.</a> J Ethol. 32 (2), 91-102 (2014).</li><li>Werner, T., Jaenike, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophilids of the Midwest and Northeast. , (2017).</li><li>Arnoult, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+and+diversification+of+fly+pigmentation+through+evolution+of+a+gene+regulatory+module.">Emergence and diversification of fly pigmentation through evolution of a gene regulatory module.</a> Science. 339 (6126), 1423-1426 (2013).</li><li>Camino, E. M., Butts, J. C., Ordway, A., Vellky, J. E., Rebeiz, M., Williams, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolutionary+origination+and+diversification+of+a+dimorphic+gene+regulatory+network+through+parallel+innovations+in+cis+and+trans.">The evolutionary origination and diversification of a dimorphic gene regulatory network through parallel innovations in cis and trans.</a> PLoS Genet. 11 (4), e1005136 (2015).</li><li>Gompel, N., Prud'homme, B., Wittkopp, P. J., Kassner, V. A., Carroll, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chance+caught+on+the+wing:+cis-regulatory+evolution+and+the+origin+of+pigment+patterns+in+Drosophila.">Chance caught on the wing: cis-regulatory evolution and the origin of pigment patterns in Drosophila.</a> Nature. 433 (7025), 481-487 (2005).</li><li>Glassford, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-option+of+an+ancestral+Hox-regulated+network+underlies+a+recently+evolved+morphological+novelty.">Co-option of an ancestral Hox-regulated network underlies a recently evolved morphological novelty.</a> Dev. Cell. 34 (5), 520-531 (2015).</li><li>Koshikawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancer+modularity+and+the+evolution+of+new+traits.">Enhancer modularity and the evolution of new traits.</a> Fly. 9 (4), 155-159 (2015).</li><li>Koshikawa, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gain+of+cis-regulatory+activities+underlies+novel+domains+of+wingless+gene+expression+in+Drosophila.">Gain of cis-regulatory activities underlies novel domains of wingless gene expression in Drosophila.</a> Proc Natl Acad Sci U S A. 112 (24), 7524-7529 (2015).</li><li>McGregor, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+evolution+through+multiple+cis-regulatory+mutations+at+a+single+gene.">Morphological evolution through multiple cis-regulatory mutations at a single gene.</a> Nature. 448 (7153), 587-590 (2007).</li><li>Tanaka, K., Barmina, O., Kopp, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+developmental+mechanisms+underlie+the+evolutionary+diversification+of+Drosophila+sex+combs.">Distinct developmental mechanisms underlie the evolutionary diversification of Drosophila sex combs.</a> Proc Natl Acad Sci U S A. 106 (12), 4764-4769 (2009).</li><li>Werner, T., Koshikawa, S., Williams, T. M., Carroll, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+novel+wing+colour+pattern+by+the+Wingless+morphogen.">Generation of a novel wing colour pattern by the Wingless morphogen.</a> Nature. 464 (7292), 1143-1148 (2010).</li><li>Wittkopp, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraspecific+polymorphism+to+interspecific+divergence:+genetics+of+pigmentation+in+Drosophila.">Intraspecific polymorphism to interspecific divergence: genetics of pigmentation in Drosophila.</a> Science. 326 (5952), 540-544 (2009).</li><li>Lawrence, P. A., Morata, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartments+in+the+wing+of+Drosophila:+a+study+of+the+engrailed+gene.">Compartments in the wing of Drosophila: a study of the engrailed gene.</a> Dev Biol. 50 (2), 321-337 (1976).</li><li>Bainbridge, S. P., Bownes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staging+the+metamorphosis+of+Drosophila+melanogaster.">Staging the metamorphosis of Drosophila melanogaster.</a> J. Embryol. Exp. Morphol. 66, 57-80 (1981).</li><li>Ashburner, M., Golic, K. G., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: A Laboratory Handbook. , (2005).</li><li>Fukutomi, Y., Matsumoto, K., Agata, K., Funayama, N., Koshikawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pupal+development+and+pigmentation+process+of+a+polka-dotted+fruit+fly,+Drosophila+guttifera+(Insecta,+Diptera).">Pupal development and pigmentation process of a polka-dotted fruit fly, Drosophila guttifera (Insecta, Diptera).</a> Dev Genes Evol. 227 (3), 171-180 (2017).</li><li>Koshikawa, S., Fukutomi, Y., Matsumoto, K., Sekimura, T., Nijhout, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+guttifera+as+a+model+system+for+unraveling+color+pattern+formation.">Drosophila guttifera as a model system for unraveling color pattern formation.</a> Diversity and evolution of butterfly wing patterns: an integrative approach. , (2017).</li><li>True, J. R., Edwards, K. A., Yamamoto, D., Carroll, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+wing+melanin+patterns+form+by+vein-dependent+elaboration+of+enzymatic+prepatterns.">Drosophila wing melanin patterns form by vein-dependent elaboration of enzymatic prepatterns.</a> Curr Biol. 9 (23), 1382-1391 (1999).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9 (7), 671-675 (2012).</li><li>Izumitani, H. F., Kusaka, Y., Koshikawa, S., Toda, M. J., Katoh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogeography+of+the+Subgenus+Drosophila+(Diptera:+Drosophilidae):+evolutionary+history+of+faunal+divergence+between+the+Old+and+the+New+Worlds.">Phylogeography of the Subgenus Drosophila (Diptera: Drosophilidae): evolutionary history of faunal divergence between the Old and the New Worlds.</a> PLoS ONE. 11 (7), e0160051 (2016).</li><li>Resh, V. H., Card&#233;, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Encyclopedia of Insects. , (2009).</li><li>DeLean, A., Munson, P. J., Rodbard, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+analysis+of+families+of+sigmoidal+curves:+application+to+bioassay,+radioligand+assay,+and+physiological+dose-response+curves.">Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiological dose-response curves.</a> Am J Physiol. 235 (2), E97-E102 (1978).</li><li>Robertson, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metamorphosis+of+Drosophila+melanogaster,+including+an+accurately+timed+account+of+the+principal+morphological+changes.">The metamorphosis of Drosophila melanogaster, including an accurately timed account of the principal morphological changes.</a> J Morphol. 59 (2), 351-399 (1936).</li><li>McKinney, M. L., McNamara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Heterochrony: the evolution of ontogeny. , (1991).</li><li>Hardie, D. C., Gregory, T. R., Hebert, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+pixels+to+picograms:+a+beginners'+guide+to+genome+quantification+by+Feulgen+image+analysis+densitometry.">From pixels to picograms: a beginners' guide to genome quantification by Feulgen image analysis densitometry.</a> J Histochem Cytochem. 50 (6), 735-749 (2002).</li><li>Koshikawa, S., Miyazaki, S., Cornette, R., Matsumoto, T., Miura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+size+of+termites+(Insecta,+Dictyoptera,+Isoptera)+and+wood+roaches+(Insecta,+Dictyoptera,+Cryptocercidae).">Genome size of termites (Insecta, Dictyoptera, Isoptera) and wood roaches (Insecta, Dictyoptera, Cryptocercidae).</a> Naturwissenschaften. 95 (9), 859-867 (2008).</li></ol>

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Wir beschreiben hier die Protokolle für die Definition von pupal Stadien entfernen der Puppenkammer für detaillierte Beobachtung, Messung der Dauer von pupal Stadien und Messung der Intensität der schwarzen Flecken auf einem Flügel in D. Guttifera. Diese Protokolle können viele Drosophila beantragt werden und im Zusammenhang mit Fliege Arten, vor allem mit Flügel Pigmentierung.
Eingehende Beobachtung und Beschreibung der detailliertere entwicklungspolitische Veranstaltu...

Einige der morphologischen Merkmale von Drosophila sind unter Arten1,2,3,4,5breit gefächert. Nähern wir uns der Frage wie morphologische Vielfalt entsteht durch den Vergleich der Mechanismen der Generation diese Morphologien. Beispiele für solche Morphologien sind Larven Trichome, adult Sex Kämme, externe Geschlechtsapparat, Abdominal-Pigmentierung und Flügel Pigmentierung6,7,8,9, 10 , 11 , 12 , 13 , 14 , 15. um morphologische Unterschiede zwischen Erwachsenen zu studieren, Beobachtung und Analyse der pupal Stadien sind wichtig, weil das Schicksal der Erwachsenen Züge sich in der späten Larvenstadien richtet und anschließende Morphogenese den pupal Zeitraum geht.
In Studien der Entwicklungsbiologie von Drosophila Melanogaster"Stunden APF" (Stunden nach pupal Bildung) ist die übliche Methode um ein Puppenstadium16anzugeben. Dieses System absoluten Zeit nach pupal Bildung beschäftigt und ist sehr praktisch für routinemäßige Experimente. Allerdings Entwicklungs-Geschwindigkeit abweichen unter Puppen und durch leichte microenvironmental, genetische und epigenetische Unterschiede betroffen sein könnten, und daher mit den gleichen absoluter Zeit nach pupal Bildung garantiert nicht, dass Puppen gleichzeitig sind Entwicklungsstadium. In vielen Fällen sind die Phasen definiert durch morphologische Merkmale für den Vergleich von mehreren Personen vorzuziehen. Vor allem, erfordert ein Vergleich zwischen den Arten präzise Inszenierung und Vergleich unter entsprechenden (homologe) Stufen.
Bainbridge und Bownes17 erkannt 20 pupal Stadien (P1 bis P15(ii)) basierend auf morphologische Merkmale von Drosophila Melanogaster Puppen. Diese Inszenierung ist die am weitesten verbreitete System der morphologischen Entwicklungs-staging-18. In einer früheren Studie führten wir pupal Inszenierung von Drosophila Guttifera , um eine Grundlage für Flügel Pigmentierung Studien19zu schaffen. D. Guttifera hat eine schwarze Polka-Dot Muster auf den Flügeln und gehört der Gattung Modell für Flügel Pigmentierung Bildung20. Obwohl wir den morphologischen Kriterien beschrieben bezeichnet die Bainbridge und Bownes Forschung17, wir direkt gemessen Bühne Dauer durch serielle Beobachtungen19, anstatt Bainbridge und Bownes Schätzung der Bühne Dauer von der beobachteten Frequenz. Hier beschreiben wir die Methode der pupal Inszenierung und Messung der Dauer von pupal Stadien der Drosophila in Fukutomi Et Al19verwendet.
Um den Entwicklungsstörungen Mechanismus der Flügel Pigmentierung zu untersuchen, müssen wir wissen, tritt im pupal oder adulten Stadien die Pigmentierung. Fukutomi Et Al. 19 quantifiziert optische dichten (ODs) der Pigmentierung während pupal und adulten Stadien durch die Bildanalyse Flügel Bilder. Die Pigmentierung von Drosophila Flügel wird gedacht, um durch Ansammlung von schwarzen Melanin21verursacht werden. Für die Quantifizierung der ODs dienten Graustufenbilder und ImageJ Software (https://imagej.nih.gov/ij/)22 . Zu erkennen und quantifizieren die Spot-spezifische Pigmentierung (ΔOD), subtrahieren wir die OD außerhalb ein Spot aus der OD innerhalb einer Stelle. Um diese Methode reproduzierbar und objektiver zu gestalten, sollten die Orte der OD-Messung über Flügel Venen als Wahrzeichen ermittelt werden. In diesem Artikel beschreiben wir ausführlich diese Methode zur Quantifizierung der Flügel Pigmentierung in Drosophila Guttifera.

<table><tbody><tr><td>&lt;em&gt;Drosophila guttifera&lt;/em&gt;</td><td>The Drosophila Species Stock Center at the U.C. San Diego</td><td>15130-1971.10</td><td>&lt;em&gt;Drosophila guttifera&lt;/em&gt;, a fruit fly species used in this article</td></tr><tr><td>Plastic vial</td><td>Hightech</td><td>MKC-30</td><td>Plastic vial, for fly stock maintenance</td></tr><tr><td>Buzz plugs vial and bottle closures for glass vials</td><td>Fisher Scientific</td><td>AS-271</td><td>Cellulose plug, for fly stock maintenance</td></tr><tr><td>White soft sugar</td><td>Mitsui Sugar</td><td>J-500g</td><td>White soft sugar, for standard cornmeal/sugar/yeast/agar food</td></tr><tr><td>Corn flour</td><td>Nippon Flour Mills</td><td>F</td><td>Corn flour, for standard cornmeal/sugar/yeast/agar food</td></tr><tr><td>Corn grits - C</td><td>Nippon Flour Mills</td><td>GC</td><td>Corn grits - C, for standard cornmeal/sugar/yeast/agar food</td></tr><tr><td>Agar powder</td><td>Matsuki Kanten Sangyo</td><td>No.602</td><td>Agar powder, for standard cornmeal/sugar/yeast/agar food</td></tr><tr><td>Dry beer yeast</td><td>Asahi Food &amp;amp; Healthcare</td><td>Y2A</td><td>Dry beer yeast, for standard cornmeal/sugar/yeast/agar food</td></tr><tr><td>Butyl p-hydroxybenzoate</td><td>Nacalai Tesque</td><td>06327-02</td><td>Butyl p-hydroxybenzoate, for standard cornmeal/sugar/yeast/agar food</td></tr><tr><td>Ethanol</td><td>Wako</td><td>057-00456</td><td>Ethanol, for standard cornmeal/sugar/yeast/agar food</td></tr><tr><td>Flat bottom microtube</td><td>Ina Optica</td><td>CF-0150</td><td>1.5 mL microtube, for collecting pupae</td></tr><tr><td>CAPSULEFUGE</td><td>Tomy</td><td>PMC-060</td><td>Mini microcentrifuge, for collecting pupae</td></tr><tr><td>Sterilized Schale NB</td><td>Sansei Medical</td><td>01-013</td><td>Plastic Petri dish (diameter 90 mm x height 15 mm)</td></tr><tr><td>Serum tube rack</td><td>Iwaki</td><td>9796-050</td><td>Used as a moist chamber, for observation of pupa</td></tr><tr><td>Corning Falcon Easy-Grip tissue culture dish</td><td>Corning</td><td>353001</td><td>Plastic Petri dish (diameter 35 mm x height 10 mm)</td></tr><tr><td>Falcon standard tissue culture dish</td><td>Corning</td><td>353002</td><td>Plastic Petri dish (diameter 60 mm x height 15 mm)</td></tr><tr><td>Push-pin</td><td>Kokuyo</td><td>51233709</td><td>Push-pin, for making pinholes on the microtube lid</td></tr><tr><td>Stereomicroscope</td><td>Olympus</td><td>SZX16</td><td>Stereomicroscope, for morphological observation</td></tr><tr><td>Digital camera</td><td>Olympus</td><td>DSE-330-A</td><td>Digital camera, for imaging</td></tr><tr><td>NICETACK double sided tape</td><td>Nichiban</td><td>NW-15SF</td><td>Double sided tape, for removing puparium</td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools</td><td>11252-20</td><td>Forceps, for removing puparium</td></tr><tr><td>Van Gogh VISUAL Paint brush</td><td>Talens Japan</td><td>GWVR-#5/0</td><td>Paint brush, for removing puparium</td></tr><tr><td>Greiner CELLSTAR 12 well cell culture plate</td><td>Merck</td><td>665-180</td><td>12-well cell culture plate, for measuring durations of pupal periods</td></tr><tr><td>NaCl</td><td>Wako</td><td>191-01665</td><td>NaCl, for PBS</td></tr><tr><td>KCl</td><td>Nacalai Tesque</td><td>285-14</td><td>KCl, for PBS</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;12H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Wako</td><td>196-02835</td><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;12H&lt;sub&gt;2&lt;/sub&gt;O, for PBS</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Nacalai Tesque</td><td>28721-55</td><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, for PBS</td></tr><tr><td>Stepped Neutral Density (ND) Filter 0.04 - 3.0</td><td>Edmund Optics</td><td>64-384</td><td>Stepped density filter, for calibration of pigmentation measurement</td></tr><tr><td>ImageJ software</td><td>NIH</td><td>1.8.0-101</td><td>ImageJ software, for measurement of intensity of black spots on a wing (https://imagej.nih.gov)</td></tr><tr><td>FINE FROST glass slide</td><td>Matsunami Glass Ind</td><td>FF-001</td><td>Glass slide, for measurement of intensity of black spots on a wing</td></tr><tr><td>Square microscope cover glass 18 x 18</td><td>Matsunami Glass Ind</td><td>C018181</td><td>Cover slip, for measurement of intensity of black spots on a wing</td></tr></tbody></table>

methods for staging pupal periods and measurement of wing pigmentation of drosophila guttifera

Vielfältige Arten von Drosophila (Fruchtfliege) bieten Möglichkeiten, die Mechanismen der Entwicklung und genetische Veränderungen für evolutionäre Veränderungen verantwortlich zu studieren. Insbesondere ist die adult-Stadium eine reiche Quelle von morphologischen Merkmalen auf interspezifischen Abgleich, einschließlich Flügel Pigmentierung Vergleich. Um Entwicklungsstörungen Unterschiede zwischen den Arten zu studieren, sind detaillierte Beobachtung und entsprechende Inszenierung für präzisen Vergleich erforderlich. Hier beschreiben wir Protokolle für die Inszenierung von pupal Perioden und Quantifizierung der Flügel Pigmentierung eine gepunktete Fruchtfliege Drosophila Guttifera. Zunächst beschreiben wir die Methode für die genaue morphologische Beobachtung und Definition von pupal Stadien basierend auf Morphologien. Diese Methode beinhaltet eine Technik zur Entfernung der Puppenkammer, ist die Radula Außenhülle der Puppe, um detaillierte Beobachtung der pupal Morphologien zu ermöglichen. Zweitens, beschreiben wir die Methode zur Messung der Dauer der definierte pupal Stadien. Schließlich beschreiben wir die Methode zur Quantifizierung der Flügel Pigmentierung basierend auf Bildanalyse mit digitalen Bildern und ImageJ-Software. Mit diesen Methoden können wir eine solide Basis für den Vergleich von Entwicklungsprozessen von Erwachsenen Züge während pupal Stadien feststellen.

Die pupal Zeitraum von D. Guttifera gliedert sich in 17 Stufen (P1 - P15(ii), Bilder von drei Vertreter, die Phasen (P1, P5 - 6, P10) gezeigt werden, Abbildung 3und alle 17 Stufen sind in Abbildung 4dargestellt). Obwohl Bainbridge und Bownes17 20 Etappen in D. Melanogastererkannt, könnten einige dieser Phasen nicht auf D. Guttiferaangewendet werden. Die Reihenfolge der beiden en...

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Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Protokolle für die Inszenierung pupal Perioden und Messung der Flügel Pigmentierung der Drosophila Guttifera werden beschrieben. Inszenierung und Quantifizierung der Pigmentierung bieten eine solide Grundlage für das Studium Entwicklungsmechanismen von Erwachsenen Züge und interspezifischen Vergleich der Eigenschaft Entwicklung ermöglichen.

Methoden zur Inszenierung Pupal Perioden und Messung der Flügel Pigmentierung der Drosophila guttifera

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary ...

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Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

11.5K Aufrufe.  Kyoto University. Protokolle für die Inszenierung pupal Perioden und Messung der Flügel Pigmentierung der Drosophila Guttifera werden beschrieben. Inszenierung und Quantifizierung der Pigmentierung bieten eine solide Grundlage für das Studium Entwicklungsmechanismen von Erwachsenen Züge und interspezifischen Vergleich der Eigenschaft Entwicklung ermöglichen.

Serie: Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells named wing imaginal discs formed during embryonic development. In the larval stages the imaginal discs grow, increasing its number of cells and forming monolayered epithelial&#160;structures. Inside the pupal case, the imaginal discs bud out and fold into bilayers along a line that becomes the future margin of the wing. During this process, the longitudinal primodia veins originate vein cells on the prospective dorsal and ventral surfaces of the wing. During the pupal stage the stripes of vein cells of each surface communicate in order to generate tight tubes; at the same time, the cross-veins begin their formation.
With the help of appropriate molecular markers, it is possible to identify the major elements composing the wing during its development. For this reason, the ability to accurately detect transcripts or proteins in this structure is critical for studying their abundance and localization related to the development process of the wing.
The procedure described here focuses on manipulating pupal wings, providing detailed instructions on how to dissect the wing during the pupal stage. The dissection of pupal tissue is more difficult to perform than their counterparts in third instar larvae. This is why this approach was developed, to obtain rapid and efficient high quality samples. Details of how to immunostain and mount these wing samples, to allow the visualization of proteins or cell components, are provided in the protocol. With little expertise it is possible to collect 8-10 high quality pupal wings in a short amount of time.

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

The ability to accurately detect transcripts or proteins in Drosophila tissues is critical for studying their abundance and localization related to the development process. Here is the description of a straightforward procedure to dissect pupal wings. These wings can be used as samples in several techniques (immunohistochemistry, PCR assay, etc.).

Eine schnelle und effiziente Methode, um Pupal Flügel von Drosophila geeignet für Immunodetections oder PCR-Assays sezieren

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells ...

Forschung

Entwicklungsbiologie

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Visualisierung von Propriozeptoren in<em&gt; Drosophila</em&gt; Larven und Puppen

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Neurowissenschaften

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

Präparation und Färbung von Drosophila Pupal Eierstöcke

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described here is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

Bildgebung und Analyse der Gewebeorientierung und Wachstumsdynamik in der Entwicklung drosophila Epithelia während der Pupal-Stadien

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila

The scaling of body parts is a central feature of animal morphology1-7. Within species, morphological traits need to be correctly proportioned to the body for the organism to function; larger individuals typically have larger body parts and smaller individuals generally have smaller body parts, such that overall body shape is maintained across a range of adult body sizes. The requirement for correct proportions means that individuals within species usually exhibit low variation in relative trait size. In contrast, relative trait size can vary dramatically among species and is a primary mechanism by which morphological diversity is produced. Over a century of comparative work has established these intra- and interspecific patterns3,4.
Perhaps the most widely used approach to describe this variation is to calculate the scaling relationship between the size of two morphological traits using the allometric equation y=bx&alpha;, where x and y are the size of the two traits, such as organ and body size8,9. This equation describes the within-group (e.g., species, population) scaling relationship between two traits as both vary in size. Log-transformation of this equation produces a simple linear equation, log(y) = log(b) + &alpha;log(x) and log-log plots of the size of different traits among individuals of the same species typically reveal linear scaling with an intercept of log(b) and a slope of &alpha;, called the 'allometric coefficient'9,10. Morphological variation among groups is described by differences in scaling relationship intercepts or slopes for a given trait pair. Consequently, variation in the parameters of the allometric equation (b and &alpha;) elegantly describes the shape variation captured in the relationship between organ and body size within and among biological groups (see 11,12).
Not all traits scale linearly with each other or with body size (e.g., 13,14) Hence, morphological scaling relationships are most informative when the data are taken from the full range of trait sizes. Here we describe how simple experimental manipulation of diet can be used to produce the full range of body size in insects. This permits an estimation of the full scaling relationship for any given pair of traits, allowing a complete description of how shape covaries with size and a robust comparison of scaling relationship parameters among biological groups. Although we focus on Drosophila, our methodology should be applicable to nearly any fully metamorphic insect.

Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila

Morphological scaling relationships capture and describe organismal shape. We present a method to measure morphological scaling relationships across the natural range of body sizes in fully metamorphic insects. Using a simple diet manipulation we increase the distribution of trait sizes, permitting the accurate description of how shape and size co-vary.

Experimentelle Manipulation von Körpergröße zu morphologischen Scaling Relationships in Estimate Drosophila</em

The scaling of body parts is a central feature of animal morphology1-7. Within species, morphological traits need to be correctly proportioned to the body ...

Biologie

<meta charset="utf8"></head><body>Erweiterte Live-Bildgebung der Morphogenese der Puppenbein von Drosophila ohne Puparium

Long-Term Live Imaging of Drosophila Pupal Leg Development After Puparium Removal

Over the past decades, significant progress has been made in understanding the mechanisms of cell fate determination. However, the process by which fate-determined cells form three-dimensional organismal shapes remains unclear. Recent advances in confocal microscopy have facilitated efforts to observe cell dynamics during development through live imaging. The Drosophila melanogaster pupa is ideal for live imaging due to its immobility, transparent pupal cuticle, and the availability of fluorescent reporter lines. A primary challenge for imaging is the puparium, the cuticle surrounding the pupa, which obstructs optical imaging. While previous methods involved either partial or complete removal of the puparium, maintaining pupal viability for extended periods after this procedure has remained challenging. Here, a simple method is presented for days-long live imaging of the Drosophila leg during the pupal stage, involving complete puparium removal. The method includes removing the puparium from a pupa adhered to double-sided tape, followed by assembling a small chamber on a glass-bottom dish to enclose the pupa and a drop of water. This setup is straightforward, reliable, and supports extended pupal survival by preventing desiccation. Long-term live imaging of the Drosophila pupa has significantly contributed to capturing how the adult leg undergoes dramatic three-dimensional structural changes over 2-3 days. These changes include the transient formation of an intriguing structure (the Parthenon-like structure) by epithelial cells, rapid tissue narrowing, joint formation, and bristle elongation. This method is applicable to the observation of various tissues and can potentially be combined with other techniques, such as optical gene induction, to advance the understanding of cell dynamics during the final shape formation of tissues in the pupal stage.

<meta charset="utf8"></head><body>Autor im Rampenlicht: Aufdeckung dynamischer Epithelstrukturen während der Entwicklung von Drosophila

Here, a protocol is presented for long-term live imaging of the Drosophila leg during the pupal stage, involving complete removal of the puparium. This protocol can also be applied to other tissues.

Langzeit-Live-Bildgebung der Entwicklung der Puppenbein von Drosophila nach Entfernung der Puppen

Over the past decades, significant progress has been made in understanding the mechanisms of cell fate determination. However, the process by which ...

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, innate immune cells perform a number of essential functions, such as fighting infection, clearing necrotic debris, and stimulating matrix deposition. In order to fully understand the diverse signaling events that regulate this immune response, it is crucial to observe the complex behaviors of (and interactions that occur between) multiple cell lineages in vivo, and in real-time, with the high spatio-temporal resolution. The optical translucency and the genetic tractability of Drosophila embryos have established Drosophila as an invaluable model to live-image and dissect fundamental aspects of inflammatory cell behavior, including mechanisms of developmental dispersal, clearance of apoptotic corpses&#160;and/or microbial pathogens, and recruitment to wounds. However, more recent work has now demonstrated that employing a much later stage in the Drosophila lifecycle &#8211; the Drosophila pupa &#8211; offers a number of distinct advantages, including improved RNAi efficiency, longer imaging periods, and significantly greater immune cell numbers. Here we describe a protocol for imaging wound repair and the associated inflammatory response at the high spatio-temporal resolution in live Drosophila pupae. To follow the dynamics of both re-epithelialization and inflammation, we use a number of specific in vivo fluorescent markers for both the epithelium and innate immune cells. We also demonstrate the effectiveness of photo-convertible fluorophores, such as Kaede, for following the specific immune cell subsets, to track their behavior as they migrate to, and resolve from, the injury site.

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene inactivation.

Langfristig In Vivo Verfolgung von entzündlichen Zelldynamik in Drosophila Puppen

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, ...

Immunologie und Infektion

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

Quantitativen Vergleich der Cis-Regulatory Element (CRE) Aktivitäten in transgenen Drosophila melanogaster</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Methoden zur Inszenierung Pupal Perioden und Messung der Flügel Pigmentierung der Drosophila guttifera

Zusammenfassung

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Bildgebung und Analyse der Gewebeorientierung und Wachstumsdynamik in der Entwicklung drosophila Epithelia während der Pupal-Stadien

Experimentelle Manipulation von Körpergröße zu morphologischen Scaling Relationships in Estimate Drosophila

Langzeit-Live-Bildgebung der Entwicklung der Puppenbein von Drosophila nach Entfernung der Puppen

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Langzeit-Live-Bildgebung der Entwicklung der Puppenbein von Drosophila nach Entfernung der Puppen

Langfristig In Vivo Verfolgung von entzündlichen Zelldynamik in Drosophila Puppen

Quantitativen Vergleich der Cis-Regulatory Element (CRE) Aktivitäten in transgenen Drosophila melanogaster