Name: protocols for visualizing steroidogenic organs and their interactive organs with immunostaining in the fruit fly drosophila melanogaster
Uploaded: 2017-04-14T14:00:00
Description: Watch this Scientific Journal Video about Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster at JoVE.com

Vi takker Reiko Kise og Tomotsune Ameku for deres tekniske støtte til dette arbeidet. Vi er også takknemlige for Kei Ito, Olga Alekseyenko, Akiko Koto, Masayuki Miura, Bloomington Drosophila Stock Center, KYOTO Stock Center (DGRC), og utviklingsstudier Hybridomproduksjon Bank for aksjer og reagenser. Dette arbeidet ble støttet med tilskudd til YSN fra JSP KAKENHI Grant Antall 16K20945, The Naito Foundation, og Inoue Science Research Award; og ved en bevilgning til RN fra MEXT KAKENHI Grant Number 16H04792.

NB: Den generelle ordningen av protokoller er vist i figur 1. 1. Disseksjon av larve Ring Gland (RG) MERK: I D. melanogaster, som hører til cyclorrhaphous Diptera, er PG innenfor et kompositt endokrint organ som kalles ring kjertel (RG, figur 2D). Siden det er ugjørlig at PG er kirurgisk separeres fra andre celletyper (omtalt senere), er et praktisk mål for å isolere et intakt og uska...

<ol>
	<li>Miller, W. L., Auchus, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Molecular+Biology,+Biochemistry,+and+Physiology+of+Human+Steroidogenesis+and+Its+Disorders.">The Molecular Biology, Biochemistry, and Physiology of Human Steroidogenesis and Its Disorders.</a> Endocr. Rev. 32 (1), 81-151 (2011).</li><li>Rousseau, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fifty+years+ago:+The+quest+for+steroid+hormone+receptors.">Fifty years ago: The quest for steroid hormone receptors.</a> Mol. Cell. Endocrinol. 375 (1), 10-13 (2013).</li><li>Gilbert, L. I., Rybczynski, R., Warren, 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=Control+and+biochemical+nature+of+the+ecdysteroidogenic+pathway.">Control and biochemical nature of the ecdysteroidogenic pathway.</a> Annu. Rev. Entomol. 47, 883-916 (2002).</li><li>Niwa, R., Niwa, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymes+for+ecdysteroid+biosynthesis:+their+biological+functions+in+insects+and+beyond.">Enzymes for ecdysteroid biosynthesis: their biological functions in insects and beyond.</a> Biosci. Biotechnol. Biochem. 78 (8), 1283-1292 (2014).</li><li>Kozlova, T., Thummel, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steroid+regulation+of+postembryonic+development+and+reproduction+in+drosophila.">Steroid regulation of postembryonic development and reproduction in drosophila.</a> Trends Endocrinol. Metab. 11 (7), 276-280 (2000).</li><li>Ishimoto, H., Kitamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+molting-roles+of+the+steroid+molting+hormone+ecdysone+in+regulation+of+memory+and+sleep+in+adult+Drosophila.">Beyond molting-roles of the steroid molting hormone ecdysone in regulation of memory and sleep in adult Drosophila.</a> Fly. 5 (3), 215-220 (2011).</li><li>Ishimoto, H., Sakai, T., Kitamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecdysone+signaling+regulates+the+formation+of+long-term+courtship+memory+in+adult+Drosophila+melanogaster.">Ecdysone signaling regulates the formation of long-term courtship memory in adult Drosophila melanogaster.</a> Proc. Natl. Acad. Sci. USA. 106 (15), 6381-6386 (2009).</li><li>Simon, A. F., Shih, C., Mack, A., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steroid+control+of+longevity+in+Drosophila+melanogaster.">Steroid control of longevity in Drosophila melanogaster.</a> Science. 299 (5611), 1407-1410 (2003).</li><li>Buszczak, M., Freeman, M. R., Carlson, J. R., Bender, M., Cooley, L., Segraves, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+Ecdysone+response+genes+govern+egg+chamber+development+during+mid-oogenesis+in+Drosophila.">a Ecdysone response genes govern egg chamber development during mid-oogenesis in Drosophila.</a> Development. 126 (20), 4581-4589 (1999).</li><li>Carney, G. E., Bender, 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+drosophila+ecdysone+receptor+(EcR)+gene+is+required+maternally+for+normal+oogenesis.">The drosophila ecdysone receptor (EcR) gene is required maternally for normal oogenesis.</a> Genetics. 154 (3), 1203-1211 (2000).</li><li>Uryu, O., Ameku, T., Niwa, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+understanding+the+role+of+ecdysteroids+in+adult+insects:+Germline+development+and+circadian+clock+in+the+fruit+fly+Drosophila+melanogaster.">Recent progress in understanding the role of ecdysteroids in adult insects: Germline development and circadian clock in the fruit fly Drosophila melanogaster.</a> Zoological Lett. 1, 32 (2015).</li><li>Ameku, T., Niwa, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mating-Induced+Increase+in+Germline+Stem+Cells+via+the+Neuroendocrine+System+in+Female+Drosophila.">Mating-Induced Increase in Germline Stem Cells via the Neuroendocrine System in Female Drosophila.</a> PLOS Genet. 12 (6), e1006123 (2016).</li><li>Danielsen, E. T., 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=A+Drosophila+Genome-Wide+Screen+Identifies+Regulators+of+Steroid+Hormone+Production+and+Developmental+Timing.">A Drosophila Genome-Wide Screen Identifies Regulators of Steroid Hormone Production and Developmental Timing.</a> Dev. Cell. 37 (6), 558-570 (2016).</li><li>Ou, Q., Zeng, J., Yamanaka, N., Brakken-Thal, C., O'Connor, M. B., King-Jones, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Insect+Prothoracic+Gland+as+a+Model+for+Steroid+Hormone+Biosynthesis+and+Regulation.">The Insect Prothoracic Gland as a Model for Steroid Hormone Biosynthesis and Regulation.</a> Cell Rep. , (2016).</li><li>Yamanaka, N., Rewitz, K. F., O'Connor, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecdysone+control+of+developmental+transitions:+lessons+from+Drosophila+research.">Ecdysone control of developmental transitions: lessons from Drosophila research.</a> Annu. Rev. Entomol. 58, 497-516 (2013).</li><li>Niwa, Y. S., Niwa, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+insect+steroid+hormone+biosynthesis+and+its+role+in+controlling+timing+of+molting+and+metamorphosis.">Transcriptional regulation of insect steroid hormone biosynthesis and its role in controlling timing of molting and metamorphosis.</a> Dev. Growth Differ. 58, 94-105 (2015).</li><li>Monastirioti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+octopamine+cell+population+residing+in+the+CNS+abdominal+ganglion+controls+ovulation+in+Drosophila+melanogaster.">Distinct octopamine cell population residing in the CNS abdominal ganglion controls ovulation in Drosophila melanogaster.</a> Dev. Biol. 264 (1), 38-49 (2003).</li><li>Siegmund, T., Korge, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innervation+of+the+ring+gland+of+Drosophila+melanogaster.">Innervation of the ring gland of Drosophila melanogaster.</a> J. Comp. Neurol. 431 (4), 481-491 (2001).</li><li>McBrayer, Z., 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=Prothoracicotropic+Hormone+Regulates+Developmental+Timing+and+Body+Size+in+Drosophila.">Prothoracicotropic Hormone Regulates Developmental Timing and Body Size in Drosophila.</a> Dev. Cell. 13 (6), 857-871 (1979).</li><li>Shimada-Niwa, Y., Niwa, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonergic+neurons+respond+to+nutrients+and+regulate+the+timing+of+steroid+hormone+biosynthesis+in+Drosophila.">Serotonergic neurons respond to nutrients and regulate the timing of steroid hormone biosynthesis in Drosophila.</a> Nat. Commun. 5, 5778 (2014).</li><li>Brady, 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+simple+technique+for+making+very+fine,+durable+dissecting+needles+by+sharpening+tungsten+wire+electrolytically.">A simple technique for making very fine, durable dissecting needles by sharpening tungsten wire electrolytically.</a> Bull World Health Organ. 32 (1), 143-144 (1965).</li><li>Abramoff, M. D., Magalh&#227;es, P. J., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+processing+with+ImageJ.">Image processing with ImageJ.</a> Biophotonics Int. 11 (7), 36-42 (2004).</li><li>Ohhara, Y., 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=Autocrine+regulation+of+ecdysone+synthesis+by+&#946;3-octopamine+receptor+in+the+prothoracic+gland+is+essential+for+Drosophila+metamorphosis.">Autocrine regulation of ecdysone synthesis by &#946;3-octopamine receptor in the prothoracic gland is essential for Drosophila metamorphosis.</a> Proc. Natl. Acad. Sci. USA. 112 (5), 1452-1457 (2015).</li><li>Gibbens, Y. Y., Warren, J. T., Gilbert, L. I., O'Connor, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroendocrine+regulation+of+Drosophila+metamorphosis+requires+TGFbeta/Activin+signaling.">Neuroendocrine regulation of Drosophila metamorphosis requires TGFbeta/Activin signaling.</a> Development. 138 (13), 2693-2703 (2011).</li><li>Parvy, J. 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=A+role+for+&#946;FTZ-F1+in+regulating+ecdysteroid+titers+during+post-embryonic+development+in+Drosophila+melanogaster.">A role for &#946;FTZ-F1 in regulating ecdysteroid titers during post-embryonic development in Drosophila melanogaster.</a> Dev. Biol. 282 (1), 84-94 (2005).</li><li>Parvy, J. -. 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=Forward+and+feedback+regulation+of+cyclic+steroid+production+in+Drosophila+melanogaster.">Forward and feedback regulation of cyclic steroid production in Drosophila melanogaster.</a> Development. 141 (20), 3955-3965 (2014).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118 (2), 401-415 (1993).</li><li>Rewitz, K. F., Yamanaka, N., Gilbert, L. I., O'Connor, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Insect+Neuropeptide+PTTH+Activates+Receptor+Tyrosine+Kinase+Torso+to+Initiate+Metamorphosis.">The Insect Neuropeptide PTTH Activates Receptor Tyrosine Kinase Torso to Initiate Metamorphosis.</a> Science. 326 (5958), 1403-1405 (2009).</li><li>Li, H. -. H., 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=A+GAL4+driver+resource+for+developmental+and+behavioral+studies+on+the+larval+CNS+of+Drosophila.">A GAL4 driver resource for developmental and behavioral studies on the larval CNS of Drosophila.</a> Cell Rep. 8 (3), 897-908 (2014).</li><li>Alekseyenko, O. V., Lee, C., Kravitz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+manipulation+of+serotonergic+neurotransmission+affects+the+escalation+of+aggression+in+adult+male+Drosophila+melanogaster.">Targeted manipulation of serotonergic neurotransmission affects the escalation of aggression in adult male Drosophila melanogaster.</a> PLOS One. 5 (5), e10806 (2010).</li><li>Domanitskaya, E., Anllo, L., Sch&#252;pbach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phantom,+a+cytochrome+P450+enzyme+essential+for+ecdysone+biosynthesis,+plays+a+critical+role+in+the+control+of+border+cell+migration+in+in+Drosophila.">Phantom, a cytochrome P450 enzyme essential for ecdysone biosynthesis, plays a critical role in the control of border cell migration in in Drosophila.</a> Dev. Biol. 386 (2), 408-418 (2014).</li><li>Song, X., Zhu, C. -. H., Doan, C., Xie, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+stem+cells+anchored+by+adherens+junctions+in+the+Drosophila+ovary+niches.">Germline stem cells anchored by adherens junctions in the Drosophila ovary niches.</a> Science. 296 (5574), 1855-1857 (2002).</li><li>Niwa, Y. S., Niwa, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+control+of+steroid+hormone+biosynthesis+during+development+in+the+fruit+fly+Drosophila+melanogaster.">Neural control of steroid hormone biosynthesis during development in the fruit fly Drosophila melanogaster.</a> Genes Genet. Syst. 89 (1), 27-34 (2014).</li><li>Yoshiyama-Yanagawa, T., 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=The+conserved+Rieske+oxygenase+DAF-36/Neverland+is+a+novel+cholesterol-metabolizing+enzyme.">The conserved Rieske oxygenase DAF-36/Neverland is a novel cholesterol-metabolizing enzyme.</a> J. Biol. Chem. 286 (29), 25756-25762 (2011).</li><li>Niwa, R., 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=Non-molting+glossy/shroud+encodes+a+short-chain+dehydrogenase/reductase+that+functions+in+the+&amp;#34;Black+Box&amp;#34;+of+the+ecdysteroid+biosynthesis+pathway.">Non-molting glossy/shroud encodes a short-chain dehydrogenase/reductase that functions in the &amp;#34;Black Box&amp;#34; of the ecdysteroid biosynthesis pathway.</a> Development. 137 (12), 1991-1999 (2010).</li><li>Komura-Kawa, T., 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=The+Drosophila+Zinc+Finger+Transcription+Factor+Ouija+Board+Controls+Ecdysteroid+Biosynthesis+through+Specific+Regulation+of+spookier.">The Drosophila Zinc Finger Transcription Factor Ouija Board Controls Ecdysteroid Biosynthesis through Specific Regulation of spookier.</a> PLOS Genet. 11 (12), e1005712 (2015).</li><li>Yamanaka, N., Marqu&#233;s, G., O'Connor, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicle-Mediated+Steroid+Hormone+Secretion+in+Drosophila+melanogaster.">Vesicle-Mediated Steroid Hormone Secretion in Drosophila melanogaster.</a> Cell. 163 (4), 907-919 (2015).</li><li>Riemensperger, T., Pech, U., Dipt, S., Fiala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+calcium+imaging+in+the+nervous+system+of+Drosophila+melanogaster.">Optical calcium imaging in the nervous system of Drosophila melanogaster.</a> BBA-Gen. Subjects. 1820 (8), 1169-1178 (2012).</li><li>Owald, D., Lin, S., Waddell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light,+heat,+action:+neural+control+of+fruit+fly+behavior.">Light, heat, action: neural control of fruit fly behavior.</a> Phil. T. Roy. Soc. B. 370 (1677), 20140211 (2015).</li></ol>

Vi studerte ekdysteroid-biosyntese og dens reguleringsmekanisme i D. melanogaster, og utviklet en protokoll for disseksjon og immunofarging. Tidspunktet for ekdysteroid-biosyntese er påvirket av omgivelses signaler gjennom neuronale innganger 33, så det er viktig å opprettholde den innervasjon av ecdysteroidogenic organene sammen med hjernen, VNC, og annet vev under dissekering. Som beskrevet ovenfor, danner D. melanogaster PG et kompleks endokrin...

I flercellede organismer, er en gruppe av celler utstyrt med et spesialisert funksjon i sin biogene aktivitet som er vesentlig for hele kroppen. For å oppfylle sine oppgaver, hvert vev eller organ uttrykker en serie av gener relatert til deres funksjoner og kommuniserer med andre vev for å organisere sin aktivitet i sammenheng med utvikling. For å karakterisere slike spesialiserte cellefunksjoner og inter-organ interaksjoner, trenger vi å angi en gruppe av celler sammen med andre typer av celler kan holdes inntakt i den flercellede arkitekturen. Et eksempel på slike spesialiserte organer er en steroidogenic organ, hvor flere biosyntetiske enzymer som medierer de konverteringstrinn fra kolesterol til aktive steroidhormoner 1. De fleste av disse enzym gener blir spesielt uttrykt i steroidogenic organer, og den biosyntetiske reaksjonsvei er strengt regulert av mange ytre stimuli via humorale innganger og neuronal innganger. En gangsyntetiserte, steroidhormoner utskilles i hemolymfe og er rettet mot en rekke vev og organer for regulering av ekspresjonen av en rekke gener 2. Derfor, induserer virkningen av et steroidhormon en systemisk respons for å opprettholde homeostase, vekst og reproduksjon. For å undersøke funksjonene av steroidhormon-biosyntese og de pleiotrope virkninger av steroidhormoner, kan Drosophila melanogaster benyttes som en egnet modell system. I løpet av larvestadiet, insekt steroidhormon, ekdysteroid, biosyntetiseres i en spesialisert endokrint organ som kalles prothoracic kjertel (PG) 3. I PG, flere ecdysteroidogenic enzymer spesifikt katalysere flere konverterings trinn fra kolesterol til ekdyson, som kontrollerer at hamskifte og metamorfose på de hensiktsmessige utviklingsstadier 4. Derfor er en dynamisk endring i ekdysteroid-titer regulertved flere signalveier som respons på miljø signaler. På den annen side, i det voksne stadium, spiller ekdysteroid viktige rolle i fysiologien, inkludert reproduksjon, søvn, hukommelse, og levetiden 5, 6, 7, 8. Det er kjent at ekdysteroid er aktivt biosyntetiseres i eggstokken, som regulerer progresjon av oogenesen 6, 7, 8, 9, 10, 11. Nylig har vi rapportert at antall kimlinje-stamceller (GSCs) påvirkes av ekdysteroid og kjønn peptid signalering som reaksjon på stimuli parring 12. Kraftige verktøy av D. melanogaster genetikk og cellebiologi, inkludert godt annotert genom informasjon, binær genekspresjonssystemer og transgene RNAi teknikker, har gjort oss i stand til å identifisere gener som er essensielle for ekdysteroid-biosyntese i PG og eggstokk 13, 14, 15. Når ecdysteroidogenic genene er identifisert, kan den transkripsjonelle regulering av disse genene og de dynamiske lokaliseringer av genprodukter bli undersøkt i den biosyntetiske reaksjonsvei 16. For dette formål, kvantitativt-revers transkripsjon-PCR, RNA in situ hybridisering, immunhistokjemi og analyse utføres. Anvendelsen av disse teknikkene omfatter en utfordrende oppgave; forseggjort disseksjon av PG eller eggstokken. Spesielt PG av bananflue er relativt mindre enn for andre insekter (f.eks silkeormen og blåse fly), så man trenger å øve på viktige ferdigheter i bananflue dissekering for prøvetaking. Videre begge ecdysteroidogenic organer motta innervasjons fra sentralnervesystemet (CNS) 17, 18, 19, 20. Således, for nøyaktig anatomiske analyser, de ecdysteroidogenic organer bør holdes intakt sammen med CNS og andre organer, for ikke å forstyrre deres nevrale forbindelser. Her gir vi protokoller for disseksjon og visualisering av steroidogenic organer i D. melanogaster. Læring disseksjon teknikk er nøkkelen utgangspunktet for disse eksperimentene. I tillegg kan man med hell merke steroidogenic organer så vel som deres interaktive organer med flere antistoffer og GAL4 driverlinjer. Å utnytte disse teknikker, materialer og genetikk, kan man studere omfattende mekanismer for steroidhormon biosyntese.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>egg collection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tissue culture dish (55 mm)</td>
			<td>AS ONE</td>
			<td>1-8549-02</td>
			<td>&#160;for grape-juice agar plates</td>
		</tr>
		<tr>
			<td>collection cup</td>
			<td>HIKARI KAGAKU</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>yeast paste</td>
			<td>Oriental dry yeast, Tokyo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100% grape juice</td>
			<td>Welch Food Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>rearing larvae</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>small vials (12ml, 40&#215;23.5 mm, PS)</td>
			<td>SARSTEDT</td>
			<td>58.487</td>
			<td></td>
		</tr>
		<tr>
			<td>disposable loop</td>
			<td>AS ONE</td>
			<td>6-488-01</td>
			<td></td>
		</tr>
		<tr>
			<td>standard fly food</td>
			<td></td>
			<td></td>
			<td>&#160;the recepi us on the website of Blooington stock center.</td>
		</tr>
		<tr>
			<td>dissection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>dissecting microscope</td>
			<td>Carl Zeiss</td>
			<td>Stemi 2000-C</td>
			<td></td>
		</tr>
		<tr>
			<td>dissecting microscope</td>
			<td>Leica</td>
			<td>S8 AP0</td>
			<td></td>
		</tr>
		<tr>
			<td>tissue culture dish (35 x 10 mm, non-treated)</td>
			<td>IWAKI</td>
			<td>1000-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard</td>
			<td>TORAY</td>
			<td></td>
			<td>coarting silicon inside dishes</td>
		</tr>
		<tr>
			<td>Terumo needle (27G, 0.40 x 19 mm)&#160;</td>
			<td>TERUMO</td>
			<td>NN-2719S</td>
			<td>A &#34;knife&#34; to cut the tissue</td>
		</tr>
		<tr>
			<td>Terumo syringe,&#12288;1ml</td>
			<td>TERUMO</td>
			<td>SS-01T</td>
			<td></td>
		</tr>
		<tr>
			<td>forceps, Inox, #5</td>
			<td>Dumont, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>insect pin (0.18 mm in diameter)</td>
			<td>Shiga Brand</td>
			<td></td>
			<td>for fillet dissection</td>
		</tr>
		<tr>
			<td>micro scissors</td>
			<td>NATSUME SEISAKUSHO CO LTD.&#160;</td>
			<td>MB-50-10</td>
			<td></td>
		</tr>
		<tr>
			<td>fixation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ultrapure water</td>
			<td>Merck Millipore</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffered saline (PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Nacalai tesque</td>
			<td>16222-65</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Nacalai tesque</td>
			<td>02890-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X100</td>
			<td>Nacalai tesque</td>
			<td>35501-15</td>
			<td></td>
		</tr>
		<tr>
			<td>microtubes (1.5 ml)</td>
			<td>INA OPTIKA</td>
			<td>CF-0150</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>As one swist mixer TM-300 (rocker)</td>
			<td>As one</td>
			<td>TM-300</td>
			<td>rocker</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>SIGMA</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr>
			<td>primary antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Sro (guinea pig), 1:1000</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-GFP (rabbit), 1:1000</td>
			<td>Molecular Probes</td>
			<td>A6455</td>
			<td>Shimada-Niwa ans Niwa, 2014</td>
		</tr>
		<tr>
			<td>anti-GFP (mouse mAb, GF200), 1:100</td>
			<td>Nakarai tesque</td>
			<td>04363-66</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-5HT (rabbit), 1:500</td>
			<td>SIGMA</td>
			<td>S5545</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Hts 1B1 (mouse)</td>
			<td>Developmental Studies Hybridoma Bank (DSHB)</td>
			<td>1B1</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-DE-cadherin (rat), 1:20</td>
			<td>DSHB</td>
			<td>DCAD2</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-nc82 (mouse), 1:50</td>
			<td>DSHB</td>
			<td>nc82</td>
			<td></td>
		</tr>
		<tr>
			<td>secondary antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td>Life Technologies</td>
			<td>A-11008</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td>Life Technologies</td>
			<td>A-11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 546 conjugate</td>
			<td>Life Technologies</td>
			<td>A-11081</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Guinea Pig IgG (H+L) Secondary Antibody, Alexa Fluor 555 conjugate</td>
			<td>Life Technologies</td>
			<td>A-21435</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 546 dye-conjugated phalloidin</td>
			<td>Life Technologies</td>
			<td>A-22283</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro slide glass</td>
			<td>Matsunami Glass Ind.,Ltd.</td>
			<td>SS7213</td>
			<td></td>
		</tr>
		<tr>
			<td>Square microscope cover glass</td>
			<td>Matsunami Glass Ind.,Ltd.</td>
			<td>C218181</td>
			<td></td>
		</tr>
		<tr>
			<td>FluorSave reagent (Mounting reagent)</td>
			<td>Calbiochem</td>
			<td>345789</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipette 1 ml (Disposable dropper)</td>
			<td>WATSON</td>
			<td>5660-222-1S</td>
			<td></td>
		</tr>
		<tr>
			<td>imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSM700 laser scanning microscope system</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>inverted Axio Observer. Z1 SP left</td>
		</tr>
		<tr>
			<td>image processing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSM700 ZEN</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>It is a special user interface based on the 64 bit Microsoft Windows7 operating system</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fly stocks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>w; GMR45C06-GAL4</td>
			<td></td>
			<td></td>
			<td>&#160;from Bloomington Drosophila Stock Center. (#46260)</td>
		</tr>
		<tr>
			<td>UAS&#8211;GFP; UAS&#8211;mCD8::GFP</td>
			<td></td>
			<td></td>
			<td>gifts from K. Ito, The University of Tokyo.</td>
		</tr>
		<tr>
			<td>w[1118]</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>w; phantom-GAL4#22/UAS-turboRFP</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>w; UAS-mCD8::GFP; TRH-GAL4</td>
			<td></td>
			<td></td>
			<td>see in Ref29, Alekseyenko, O. V, Lee, C. &#38; Kravitz, E. A.(2010)</td>
		</tr>
		<tr>
			<td>w; UAS-mCD8::GFP</td>
			<td></td>
			<td></td>
			<td>&#160;from Bloomington Drosophila Stock Center. (#32188)</td>
		</tr>
		<tr>
			<td>yw;; nSyb-GAL4</td>
			<td></td>
			<td></td>
			<td>&#160;from Bloomington Drosophila Stock Center. (#51941)</td>
		</tr>
	</tbody>
</table>

protocols for visualizing steroidogenic organs and their interactive organs with immunostaining in the fruit fly drosophila melanogaster

I flercellede organismer, er en liten gruppe av celler utstyrt med et spesialisert funksjon i sin biogene aktivitet som induserer en systemisk respons til vekst og reproduksjon. I insekter, larve prothoracic kjertel (PG) og den voksne kvinnelige eggstokk spille viktige roller i biosynthesizing de viktigste steroidhormoner som kalles ekdysteroider. Disse ecdysteroidogenic organene er innervert fra nervesystemet, gjennom hvilken tidspunktet for biosyntese påvirkes av miljø signaler. Her beskriver vi en protokoll for å visualisere ecdysteroidogenic organer og deres interaktive organer i larver og voksne av bananflue Drosophila melanogaster, som gir en passende modellsystem for å studere steroid hormon biosyntese og dens reguleringsmekanisme. Dyktige disseksjon tillater oss å opprettholde posisjonene til ecdysteroidogenic organer og deres interaktive organer, inkludert hjernen, det ventrale nervesystemet og andre vev. Farging med enntibodies mot ecdysteroidogenic enzymene, sammen med transgene fluorescens proteiner som drives av vevs-spesifikke promotorer er tilgjengelige for å merke ecdysteroidogenic celler. Videre kan innervations av ecdysteroidogenic organer også bli merket spesifikke antistoffer eller en samling av GAL4 sjåfør forskjellige typer av nerveceller. Derfor kan ecdysteroidogenic organer og deres nevrale forbindelser bli visualisert samtidig av immunfarging og transgene teknikker. Til slutt, vil vi beskrive hvordan å visualisere kimlinje stamceller, hvis spredning og vedlikeholds styres av ekdysteroider. Denne fremgangsmåten bidrar til omfattende forståelse av steroidhormon-biosyntese og dens neuronal reguleringsmekanisme.

Vi brukte de ovennevnte protokoller for å visualisere steroidogenic organer og deres interaktive organer i D. melanogaster larver og voksne kvinner. Det totale skjema av protokoller er vist i figur 1. RG, inkludert PG (figur 2D), er mindre og mer gjennomsiktige enn hjernen og er plassert ved den fremre-ryggsiden av hjernen (figur 2A-C og 3A-E). Å merke PG-celler, har flere grupper generert forskjellige typer av antistoffer mot ecdysteroidogenic enzymer <s...

Watch this Scientific Journal Video about Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster at JoVE.com

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

Vi beskriver en protokoll for disseksjon, fiksering og immunofarging av steroidogenic organer i Drosophila-larver og voksne kvinner for å studere steroid hormon biosyntese og dens reguleringsmekanisme. I tillegg til steroidogenic organer, visual vi innervasjon av steroidogenic organer samt steroidogenic målceller slik som kimlinje-stamceller.

Protokoller for Visualisering Steroidogenic organer og deres Interaktiv organer med Farging i bananflue<em&gt; Drosophila melanogaster</em

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

Research

JoVE Journal

Developmental Biology

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Analyse av blodcellepopulasjoner i<em&gt; Drosophila melanogaster</em&gt; Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

Oppdragelse bananflue<em&gt; Drosophila melanogaster</em&gt; Under Axenic og gnotobiotiske betingelser

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is ...

Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

Visualisere Interrenal Steroidogenic Tissue og dens Vaskulær mikromiljøet i Sebrafisk

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying ...

High-resolution Episcopic Microscopy (HREM) - Simple and Robust Protocols for Processing and Visualizing Organic Materials

High-resolution Episcopic Microscopy HREM - Simple and Robust Protocols for Processing and Visualizing Organic Materials

Høyoppløselig episkopisk mikroskopi (HREM) - Enkle og robuste protokoller for behandling og visualisering av organiske materialer

We provide simple protocols for generating digital volume data with the high-resolution episcopic microscopy (HREM) method. HREM is capable of imaging ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Disseksjon og Immunofluorescent flekker av sopp kropp og Photoreceptor Neurons i voksen Drosophila melanogaster hjernen

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Hele-mount Clearing og flekker av Arabidopsis blomst organer og Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Celle aggregering analyser evaluere bindingen Drosophila hakket med Trans-ligander og dens hemming av Cis-ligander

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Tredimensjonal gjengivelse og analyse av Immunolabeled, avklart menneskelige Placental Villous vaskulære nettverk

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

Tredimensjonale Organotypic kulturer av Vestibular og auditiv Sanseorganer

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

Visualisere Node og Notochordal Plate i Gastrulating Mouse embryoer skanning elektronmikroskop og hele Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...