We thank Matthew O'Connell, Maryam Jahanshahi, and Andreas Jenny for assistance. We thank Istv&#225;n And&#243; for plasmatocyte-specific antibodies, Utpal Banerjee for dome-meso-EBFP2 flies, Julian Martinez-Agosto for antp&gt;GFP flies, and Michael O'Connor for ptth and ptth&gt;grim flies. These methods were developed with support by the Kimmel Foundation, the Leukemia &amp; Lymphoma Society, NIH/NCI R01CA140451, NSF 1257939, DOD/NFRP W81XWH-14-1-0059, and NIH/NCI T32CA078207.

<ol>
	<li>Evans, C. J., Hartenstein, V., Banerjee, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thicker+than+blood:+conserved+mechanisms+in+Drosophila+and+vertebrate+hematopoiesis.">Thicker than blood: conserved mechanisms in Drosophila and vertebrate hematopoiesis.</a> Dev Cell. 5 (5), 673-690 (2003).</li><li>Crozatier, M., Meister, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+haematopoiesis.">Drosophila haematopoiesis.</a> Cell Microbiol. 9 (5), 1117-1126 (2007).</li><li>Crozatier, M., Vincent, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila:+a+model+for+studying+genetic+and+molecular+aspects+of+haematopoiesis+and+associated+leukaemias.">Drosophila: a model for studying genetic and molecular aspects of haematopoiesis and associated leukaemias.</a> Dis Model Mech. 4 (4), 439-445 (2011).</li><li>Gold, K. S., Bruckner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+for+the+two+myeloid+blood+cell+systems+in+vertebrates.">Drosophila as a model for the two myeloid blood cell systems in vertebrates.</a> Exp Hematol. 42 (8), 717-727 (2014).</li><li>Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+cells+and+blood+cell+development+in+the+animal+kingdom.">Blood cells and blood cell development in the animal kingdom.</a> Annu Rev Cell Dev Biol. 22, 677-712 (2006).</li><li>Ghosh, S., Singh, A., Mandal, S., Mandal, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+hematopoietic+hubs+in+Drosophila+adults+generate+hemocytes+and+contribute+to+immune+response.">Active hematopoietic hubs in Drosophila adults generate hemocytes and contribute to immune response.</a> Dev Cell. 33 (4), 478-488 (2015).</li><li>Leitao, A. B., Sucena, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+sessile+hemocyte+clusters+are+true+hematopoietic+tissues+that+regulate+larval+blood+cell+differentiation.">Drosophila sessile hemocyte clusters are true hematopoietic tissues that regulate larval blood cell differentiation.</a> Elife. 4, (2015).</li><li>Makhijani, K., Alexander, B., Tanaka, T., Rulifson, E., Bruckner, 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+peripheral+nervous+system+supports+blood+cell+homing+and+survival+in+the+Drosophila+larva.">The peripheral nervous system supports blood cell homing and survival in the Drosophila larva.</a> Development. 138 (24), 5379-5391 (2011).</li><li>Crozatier, M., Ubeda, J. M., Vincent, A., Meister, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+immune+response+to+parasitization+in+Drosophila+requires+the+EBF+orthologue+collier.">Cellular immune response to parasitization in Drosophila requires the EBF orthologue collier.</a> PLoS Biol. 2 (8), 196 (2004).</li><li>Markus, 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=Sessile+hemocytes+as+a+hematopoietic+compartment+in+Drosophila+melanogaster.">Sessile hemocytes as a hematopoietic compartment in Drosophila melanogaster.</a> Proc Natl Acad Sci U S A. 106 (12), 4805-4809 (2009).</li><li>Minakhina, S., Steward, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanotic+mutants+in+Drosophila:+pathways+and+phenotypes.">Melanotic mutants in Drosophila: pathways and phenotypes.</a> Genetics. 174 (1), 253-263 (2006).</li><li>Bina, S., Wright, V. M., Fisher, K. H., Milo, M., Zeidler, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+targets+of+Drosophila+JAK/STAT+pathway+signalling+as+effectors+of+haematopoietic+tumour+formation.">Transcriptional targets of Drosophila JAK/STAT pathway signalling as effectors of haematopoietic tumour formation.</a> EMBO Rep. 11 (3), 201-207 (2010).</li><li>Avet-Rochex, A., 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=An+in+vivo+RNA+interference+screen+identifies+gene+networks+controlling+Drosophila+melanogaster+blood+cell+homeostasis.">An in vivo RNA interference screen identifies gene networks controlling Drosophila melanogaster blood cell homeostasis.</a> BMC Dev Biol. 10, 65 (2010).</li><li>Rodriguez, A., 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=Identification+of+immune+system+and+response+genes,+and+novel+mutations+causing+melanotic+tumor+formation+in+Drosophila+melanogaster.">Identification of immune system and response genes, and novel mutations causing melanotic tumor formation in Drosophila melanogaster.</a> Genetics. 143 (2), 929-940 (1996).</li><li>Mandal, L., Banerjee, U., Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+fruit+fly+hemangioblast+and+similarities+between+lymph-gland+hematopoiesis+in+fruit+fly+and+mammal+aorta-gonadal-mesonephros+mesoderm.">Evidence for a fruit fly hemangioblast and similarities between lymph-gland hematopoiesis in fruit fly and mammal aorta-gonadal-mesonephros mesoderm.</a> Nat Genet. 36 (9), 1019-1023 (2004).</li><li>Grigorian, M., Mandal, L., Hakimi, M., Ortiz, I., Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+convergence+of+Notch+and+MAPK+signaling+specifies+the+blood+progenitor+fate+in+the+Drosophila+mesoderm.">The convergence of Notch and MAPK signaling specifies the blood progenitor fate in the Drosophila mesoderm.</a> Dev Biol. 353 (1), 105-118 (2011).</li><li>Lebestky, T., Jung, S. H., Banerjee, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Serrate-expressing+signaling+center+controls+Drosophila+hematopoiesis.">A Serrate-expressing signaling center controls Drosophila hematopoiesis.</a> Genes Dev. 17 (3), 348-353 (2003).</li><li>Krzemien, 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=Control+of+blood+cell+homeostasis+in+Drosophila+larvae+by+the+posterior+signalling+centre.">Control of blood cell homeostasis in Drosophila larvae by the posterior signalling centre.</a> Nature. 446 (7133), 325-328 (2007).</li><li>Mandal, L., Martinez-Agosto, J. A., Evans, C. J., Hartenstein, V., Banerjee, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Hedgehog-+and+Antennapedia-dependent+niche+maintains+Drosophila+haematopoietic+precursors.">A Hedgehog- and Antennapedia-dependent niche maintains Drosophila haematopoietic precursors.</a> Nature. 446 (7133), 320-324 (2007).</li><li>Benmimoun, B., Polesello, C., Haenlin, M., Waltzer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+EBF+transcription+factor+Collier+directly+promotes+Drosophila+blood+cell+progenitor+maintenance+independently+of+the+niche.">The EBF transcription factor Collier directly promotes Drosophila blood cell progenitor maintenance independently of the niche.</a> Proc Natl Acad Sci U S A. 112 (29), 9052-9057 (2015).</li><li>Sinenko, S. A., Mandal, L., Martinez-Agosto, J. A., Banerjee, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+role+of+wingless+signaling+in+stem-like+hematopoietic+precursor+maintenance+in+Drosophila.">Dual role of wingless signaling in stem-like hematopoietic precursor maintenance in Drosophila.</a> Dev Cell. 16 (5), 756-763 (2009).</li><li>Pennetier, D., 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=Size+control+of+the+Drosophila+hematopoietic+niche+by+bone+morphogenetic+protein+signaling+reveals+parallels+with+mammals.">Size control of the Drosophila hematopoietic niche by bone morphogenetic protein signaling reveals parallels with mammals.</a> Proc Natl Acad Sci U S A. 109 (9), 3389-3394 (2012).</li><li>Dragojlovic-Munther, M., Martinez-Agosto, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix-modulated+Heartless+signaling+in+Drosophila+blood+progenitors+regulates+their+differentiation+via+a+Ras/ETS/FOG+pathway+and+target+of+rapamycin+function.">Extracellular matrix-modulated Heartless signaling in Drosophila blood progenitors regulates their differentiation via a Ras/ETS/FOG pathway and target of rapamycin function.</a> Dev Biol. 384 (2), 313-330 (2013).</li><li>Ferguson, G. B., Martinez-Agosto, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yorkie+and+Scalloped+signaling+regulates+Notch-dependent+lineage+specification+during+Drosophila+hematopoiesis.">Yorkie and Scalloped signaling regulates Notch-dependent lineage specification during Drosophila hematopoiesis.</a> Curr Biol. 24 (22), 2665-2672 (2014).</li><li>Milton, C. C., 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+Hippo+pathway+regulates+hematopoiesis+in+Drosophila+melanogaster.">The Hippo pathway regulates hematopoiesis in Drosophila melanogaster.</a> Curr Biol. 24 (22), 2673-2680 (2014).</li><li>Evans, C. J., Liu, T., Banerjee, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+hematopoiesis:+Markers+and+methods+for+molecular+genetic+analysis.">Drosophila hematopoiesis: Markers and methods for molecular genetic analysis.</a> Methods. 68 (1), 242-251 (2014).</li><li>Neyen, C., Bretscher, A. J., Binggeli, O., Lemaitre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+study+Drosophila+immunity.">Methods to study Drosophila immunity.</a> Methods. 68 (1), 116-128 (2014).</li><li>Small, C., Paddibhatla, I., Rajwani, R., Govind, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+introduction+to+parasitic+wasps+of+Drosophila+and+the+antiparasite+immune+response.">An introduction to parasitic wasps of Drosophila and the antiparasite immune response.</a> J Vis Exp. (63), e3347 (2012).</li><li>Petraki, S., Alexander, B., Bruckner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+Blood+Cell+Populations+of+the+Drosophila+melanogaster+Larva.">Assaying Blood Cell Populations of the Drosophila melanogaster Larva.</a> J Vis Exp. (105), (2015).</li><li>Rizki, M. T. M., Rizki, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+significance+of+the+crystal+cells+in+the+larva+of+Drosophila+mekmogaster.">Functional significance of the crystal cells in the larva of Drosophila mekmogaster.</a> Journal of Biophysical and Biochemical Cytology. 5, 235-240 (1959).</li><li>Kurucz, E., 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=Definition+of+Drosophila+hemocyte+subsets+by+cell-type+specific+antigens.">Definition of Drosophila hemocyte subsets by cell-type specific antigens.</a> Acta Biol Hung. 58, 95-111 (2007).</li><li>Jung, S. H., Evans, C. J., Uemura, C., Banerjee, U. <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+lymph+gland+as+a+developmental+model+of+hematopoiesis.">The Drosophila lymph gland as a developmental model of hematopoiesis.</a> Development. 132 (11), 2521-2533 (2005).</li><li>Krzemien, J., Crozatier, M., Vincent, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ontogeny+of+the+Drosophila+larval+hematopoietic+organ,+hemocyte+homeostasis+and+the+dedicated+cellular+immune+response+to+parasitism.">Ontogeny of the Drosophila larval hematopoietic organ, hemocyte homeostasis and the dedicated cellular immune response to parasitism.</a> Int J Dev Biol. 54 (6-7), 1117-1125 (2010).</li><li>Rizki, T. M., Rizki, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+Larval+Hemocytes+of+Drosophila-Melanogaster.">Properties of the Larval Hemocytes of Drosophila-Melanogaster.</a> Experientia. 36 (10), 1223-1226 (1980).</li><li>Lanot, R., Zachary, D., Holder, F., Meister, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postembryonic+hematopoiesis+in+Drosophila.">Postembryonic hematopoiesis in Drosophila.</a> Dev Biol. 230 (2), 243-257 (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 (2007).</li><li>Reimels, T. A., Pfleger, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Rabex-5+restricts+Notch+activity+in+hematopoietic+cells+and+maintains+hematopoietic+homeostasis.">Drosophila Rabex-5 restricts Notch activity in hematopoietic cells and maintains hematopoietic homeostasis.</a> J Cell Sci. 128 (24), 4512-4525 (2015).</li></ol>

החוקרים מצהירים כי אין להם אינטרסים מתחרים או פיננסיים.

עם השינוי גנטי או סביבתי, ארבע השיטות שתוארו כאן ניתן להשתמש בנפרד או בשילוב לנתח תהליכים שונים במהלך hematopoiesis כגון איתות, הישרדות, שגשוג, והבחנת hematopoiesis תסיסנית היא תהליך דינאמי.; מספר hemocytes לכל חיה מגדיל 35 והביטוי מבנה הגן של בלוטת הלימפה משנה 32 במהלך ?...

המנגנונים המורכבים המסדירים את גורמי שעתוק מסלולי איתות כי לתאם את הפיתוח של מערכת hematopoietic וכי תקלה במחלות המטולוגיות להישאר ממעטים להבין. גורמי שעתוק אלה מסלולי איתות, כמו גם הוויסות שלהם, הם שמורים ביותר בין תסיסנית יונקי hematopoiesis 1-5. לכן מערכת hematopoietic תסיסנית מייצגת מודל גנטי מעולה להגדיר את המנגנונים המולקולריים השולטים hematopoiesis ומחלות המטולוגיות בסיסיות. בדומה ליונקים, תסיסנית ליצור תאי דם, המכונה hemocytes, ב במרחב ובזמן שלבים ברורים של hematopoiesis. באופן מסורתי, hematopoiesis תסיסנית נחשב להיות מוגבל שלבי ביניים mesoderm העוברי בבלוטת הלימפה הזחל. מחקרים שנעשו לאחרונה לספק ראיות כי hematopoiesis מתרחשת גם CLU נייחים הזחלsters והן מבוגר בטן 6-8. כל שלבי hematopoietic לייצר שני סוגים של hemocytes הבוגרת: plasmatocytes ותאי קריסטל. Plasmatocytes הם תאים דמויי מקרופאג מעורב phagocytosis, חסינות מולדת, וריפוי פצעים. תאים קריסטל מכילים פרו-phenoloxidases הנדרשים melanization, תגובה המשמשת תגובות חיסוניות חרקים וריפוי פצעים. Hematopoiesis זחל יכול ליצור סוג hemocyte בוגרת שלישי, שנקרא lamellocyte, בתגובה לאתגרי חיסון מסוימים כגון זיהום צרעה טפיל 9,10. Lamellocytes גדול, תאים חסידים המתפקדים, בשיתוף עם plasmatocytes ותאי קריסטל, לתמצת ולנטרל ביצי צרעה הניחו זחלים תסיסנית. בהעדר parasitization, lamellocytes לא נמצא זחלי wild-type. המוני Melanotic להידמות ביצי צרעת melanized, כמוסות; רבי זנים תסיסנית מוטציה לפתח המוני melanotic בהעדר parasitization. הנוכחות של lamellocytes ו / או המוני melanotic יכול להצביע על ליקויי hematopoietic. למעשה, את הפנוטיפ המוני melanotic נעשה שימוש כדי לזהות גנים ושבילים מעורבים hematopoiesis 11-14. מערכת hematopoietic הזחל הוא נרחב ביותר למד עד כה. היא מורכבת hemocytes במחזור hemolymph, אשכולות hemocyte נייחים בדוגמת תחת לציפורן, ו hemocytes המתגוררים בלוטת הלימפה. בלוטת הלימפה היא סדרה של אונות הבילטרליים מצורפות הכלי הגבה. כל אונה העיקרית של בלוטת הלימפה מחולקת לשלושה אזורים עיקריים. האזור החיצוני ידוע כאזור קליפת המוח ומכיל התבגרות hemocytes. האזור הפנימי ביותר נקרא האזור מדולרי והיא מורכבת מבשרי hemocyte שקטים. האזור השלישי, במרכז איתות האחורי, הוא קבוצה קטנה של תאים בבסיס של בלוטת הלימפה אשר פועלת נישה דמוית תא גזע. עבודה מוקדמת הוקמה פונקציות קריטיות עבור Notch 15-18 </sup&gt;, קיפוד 19,20, JAK-STAT 18, ו כנפי 21 פעילות להסדיר התפתחות בלוטת הלימפה זחל. מחקרים מאוחרים יותר הראו כי BMP 22, FGF-ראס 23, ו היפו 24,25 איתות גם לתפקד בתוך בלוטת הלימפה הזחל. ארבעה מבחני hematopoietic הזחל המפורטים כאן לתאר 1) מדידת מחזורי ריכוז hemocyte, מוגדר כמספר של תאים ליחידת נפח, 2) בידוד ותיקון במחזור hemocytes אימונוהיסטוכימיה, 3) חזותי בתאי קריסטל in vivo, ו -4) לנתח, לתקן, ואת הרכבה הלימפה בלוטות אימונוהיסטוכימיה. מבחנים אלה יכולים לשמש readouts hematopoietic להעריך את הפונקציות והתקנות של מסלולי איתות במערכת hematopoietic הזחל. בעוד שיטות אלה שמשו בעבר בתחום, תיעוד חזותי של מבחנים אלה החל רק לאחרונה 8,26-30. מספר פרסומים שצוטטו כאן הם לעזרהמשאבי פול המתארים בשיטות דומות וסמני hematopoietic 26,31-33. בנוסף, Trol וויקינג הם סמנים השימושיים של קרום במרתף בלוטת הלימפה.

<table><tbody><tr><td>PBS tablets</td><td>MP Biomedicals</td><td>2810305</td><td></td></tr><tr><td>dissecting dish</td><td>Corning</td><td>7220-85</td><td></td></tr><tr><td>microcentrifuge tube</td><td>Denville</td><td>C2170</td><td></td></tr><tr><td>silicone dissecting pad, made from Sylgard 184 kit</td><td>Krayden (distributed through Fisher)</td><td>NC9644388 (Fisher catalog number)</td><td>Made in petri dish by mixing components of Sylgard elastomer kit according to manufacturer instructions.</td></tr><tr><td>stereomicroscope</td><td>Morrell Instruments (Nikon distributor)</td><td>mna42000, mma36300</td><td>Nikon models SMZ1000 and SMZ645</td></tr><tr><td>tissue wipe</td><td>VWR</td><td>82003-820</td><td></td></tr><tr><td>forceps</td><td>Electron Microscopy Sciences</td><td>72700-DZ</td><td></td></tr><tr><td>p200 pipette</td><td>Eppendorf</td><td>3120000054</td><td></td></tr><tr><td>Countess Automated Cell Counter</td><td>Invitrogen</td><td>C10227</td><td></td></tr><tr><td>Countess cell counting chamber slides</td><td>Invitrogen</td><td>C10283</td><td></td></tr><tr><td>hemocytometer</td><td>Hausser Scientific</td><td>3200</td><td></td></tr><tr><td>trypan blue stain</td><td>Life Technologies</td><td>T10282</td><td></td></tr><tr><td>formaldehyde</td><td>Fisher</td><td>BP531-500</td><td></td></tr><tr><td>Triton</td><td>Fisher</td><td>BP151-500</td><td></td></tr><tr><td>Tween 20</td><td>Fisher</td><td>BP337-500</td><td></td></tr><tr><td>bovine serum albumin</td><td>Rocky Mountain Biologicals</td><td>BSA-BSH-01K</td><td></td></tr><tr><td>normal goat serum</td><td>Sigma</td><td>G9023-10ML</td><td></td></tr><tr><td>normal donkey serum</td><td>Sigma</td><td>D9663-10ML</td><td></td></tr><tr><td>200 proof ethanol</td><td>VWR</td><td>V1001</td><td></td></tr><tr><td>N-propyl gallate</td><td>MP Biomedicals</td><td>102747</td><td></td></tr><tr><td>glycerol</td><td>VWR</td><td>EM-4750</td><td></td></tr><tr><td>DAPI (4&amp;rsquo;,6-diamidino-2-phenylindole)</td><td>Fisher</td><td>62248</td><td></td></tr><tr><td>6-well plate</td><td>Corning</td><td>351146</td><td></td></tr><tr><td>12-well plate</td><td>Corning</td><td>351143</td><td></td></tr><tr><td>microscope cover glass, 22 mm square</td><td>Fisher</td><td>12-544-10</td><td></td></tr><tr><td>microscope cover glass, 18 mm circular</td><td>Fisher</td><td>12-545-100</td><td></td></tr><tr><td>glass microscope slides</td><td>Fisher</td><td>22-034-980</td><td></td></tr><tr><td>thermal cycler</td><td>Eppendorf</td><td>E950010037</td><td>Mastercycler EP Gradient S</td></tr><tr><td>PCR tubes</td><td>USA Scientific</td><td>1402-2700</td><td></td></tr><tr><td>24-well plate</td><td>Corning</td><td>351147</td><td></td></tr><tr><td>disposable transfer pipet</td><td>Fisher</td><td>13-711-9AM</td><td></td></tr><tr><td>fluorescence microscope</td><td>Zeiss</td><td></td><td>Axio Imager.Z1</td></tr></tbody></table>

methods to examine the lymph gland and hemocytes in drosophila larvae

קבלות רבות קיימות בין תסיסנית ומערכות hematopoietic יונקות, למרות תסיסנית חסרות את שושלת הלימפה מאפיינות חסינות אדפטיבית יונקת. תסיסנית hematopoiesis יונק להתרחש שלבים ברורים במרחב ובזמן כדי לייצר כמה שושלות תאי דם. שני המערכות לשמור מאגרים של אבות תא דם שבה להרחיב או להחליף שושלות בוגרות. מערכת hematopoietic מאפשרת תסיסנית ויונק להגיב ולהתאים לאתגרים חיסוניים. חשוב לציין, הרגולטורים תעתיק מסלולי איתות השולטים הדור, תחזוקה, והתפקוד של מערכת hematopoietic שמורים מפני זבובים ליונקים. דמיון אלה מאפשרים תסיסנית לשמש לפיתוח ומחלות hematopoietic מודל גנטי. כאן אנו מבחני פרט לבחון את מערכת hematopoietic של הזחלים תסיסנית. בבפרט, אנו המתאר שיטות למדוד במספרי תא דם וריכוז, לדמיין שושלת בוגרת ספציפית in vivo, ולבצע אימונוהיסטוכימיה על תאי דם שבמחזור באיבר hematopoietic. מבחנים אלה יכולים לגלות שינויים בביטוי גנים ואת תהליכים תאיים כולל איתות, הישרדות, התפשטות, בידול יכולים לשמש כדי לחקור מגוון רחב של שאלות הנוגעות hematopoiesis. בשילוב עם הכלים הגנטיים הזמינים תסיסנית, יכולים לשמש מבחנים אלה כדי להעריך את מערכת hematopoietic על שינויים גנטיים מוגדרים. אמנם לא התווה במיוחד כאן, מבחנים אלה יכולים לשמש גם כדי לבחון את השפעת שינויים סביבתיים, כגון זיהום או דיאטה, על מערכת hematopoietic.

במחזור ריכוז Hemocyte מספרים Hemocyte ותגבר במהלך התפתחות הזחל 35. כדי להמחיש כי שיטה זו מזהה הבדלים במספרי hemocyte וריכוז, ללא קשר לסיבה הביולוגית, מדדנו ריכוזי hemocyte של זחלים מתעכבים ולא להתעכב. פסד ש?...

Watch this Scientific Journal Video about Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae at JoVE.com

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

תסיסנית ומערכות hematopoietic יונקים חולקים הרבה מאפיינים משותפים, מה שהופך תסיסנית מודל גנטי אטרקטיבי ללמוד hematopoiesis. כאן אנו מדגימים לנתיחת הרכבה של איבר hematopoietic הגדול זחל אימונוהיסטוכימיה. כמו כן, אנו מתארים שיטות Assay שונים תאי hematopoietic זחל כולל hemocytes במחזור ותאי קריסטל נייחים.

שיטות לבדיקת בלוטת לימפה hemocytes ב<em&gt; תסיסנית</em&gt; זחלים

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

methods-to-examine-the-lymph-gland-and-hemocytes-in-drosophila-larvae

Research

JoVE Journal

Developmental Biology

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

12.7K Views.  The Icahn School of Medicine at Mount Sinai.  תסיסנית ומערכות hematopoietic יונקים חולקים הרבה מאפיינים משותפים, מה שהופך תסיסנית מודל גנטי אטרקטיבי ללמוד hematopoiesis. כאן אנו מדגימים לנתיחת הרכבה של איבר hematopoietic הגדול זחל אימונוהיסטוכימיה. כמו כן, אנו מתארים שיטות Assay שונים תאי hematopoieti...

Video: Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage.&#160;HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

אוכלוסיות תאי דם Assaying של<em&gt; תסיסנית melanogaster</em&gt; זחל

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

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

מבוא של צרעות טפיליות<em&gt; תסיסנית</em&gt; ואת התגובה החיסונית Antiparasite

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Immunology and Infection

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

ניתוח פונקציונלי של מעגל האכלת הזחל ב דרוזופילה</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

Neuroscience

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

הדמיה חיה של תסיסנית melanogaster Hemocyte נדידות עובריים

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Biology

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

החילוץ של Hemocytes של הזחלים דרוזופילה melanogaster זיהום מיקרוביאלי וניתוח

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Assessing the Age-Specific Phagocytic Ability of Adult Drosophila melanogaster Hemocytes using an In Vivo Phagocytosis Assay

Phagocytosis is an essential function of the innate immune response. This process is carried out by phagocytic hemocytes whose primary function is to recognize a wide range of particles and destroy microbial pathogens. As organisms age, this process begins to decline, yet little is known about the underlying mechanisms or the genetic basis of immunosenescence. Here, an injection based in vivo phagocytosis assay is used to assess age related changes in different aspects of phagocytosis, such as binding, engulfment, and degradation of internalized particles, by quantifying phagocytic events in hemocytes in adult Drosophila. Drosophila melanogaster has become an ideal model to investigate age related changes in innate immune function for many reasons. For one, many genetic components and functions of the innate immune response, including phagocytosis, are evolutionarily conserved between Drosophila and mammals. Because of that, results obtained from using this protocol are likely to be widely relevant to understanding the age related changes in immune function in a variety of organisms. Additionally, we note that this method provides quantitative estimates of hemocyte phagocytic ability, which could be useful for a variety of research topics, and need not be limited to studies of aging.

Assessing the Age-Specific Phagocytic Ability of Adult Drosophila melanogaster Hemocytes using an In Vivo Phagocytosis Assay

This protocol describes an in vivo assay of phagocytosis used to assess and quantify the ability of young and aged Drosophila melanogaster hemocytes to phagocytose bacteria.

הערכת היכולת הייחודית ביותר לגיל מיוחד למבוגרים דרוזופילה מלאנוטציטים באמצעות שיטת בVivo פגוציטוזה

Phagocytosis is an essential function of the innate immune response. This process is carried out by phagocytic hemocytes whose primary function is to ...

Drosophila Larval IHC

Immunohistochemistry (IHC) is a technique used to visualize the presence and location of proteins within tissues. Drosophila larvae are particularly amenable to IHC because of the ease with which they can be processed for staining. Additionally, the larvae are transparent, meaning that some tissues can be visualized without the need for dissection.
In IHC, proteins are ultimately detected with antibodies that specifically bind to &ldquo;epitopes&rdquo; within the protein of interest. In order to preserve these epitopes, tissues must be fixed prior to staining. Furthermore, in order for antibodies to penetrate membranes, cells must be permeabilized with detergents. This video article offers a detailed view of the reagents, tools, and procedures necessary for the staining of dissected larval tissues, including fixation, blocking, and staining steps. Also featured is a demonstration of tissue mounting techniques for fluorescence microscopy. Finally, examples of the broad range of applications for these techniques (and some variations upon them) are provided.

Drosophila melanogaster Larval Immunohistochemistry IHC

דרוסופילים Larval IHC

Immunohistochemistry (IHC) is a technique used to visualize the presence and location of proteins within tissues. Drosophila larvae are particularly ...

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Basic Biology

Biology I: yeast, Drosophila and C. elegans

Cuticle Disruption: A Method to Collect Hemolymph from Drosophila Larvae

Source: Reimels, T. A. and Pfleger, C. M. <a href="https://www.jove.com/video/54544/" target="_blank">Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae</a>. J. Vis. Exp. (2016).
The Drosophila hemolymph serves as both blood and interstitial fluid, circulating throughout the body during the larva and adult stages. This video describes a protocol to collect larval hemolymph in order to quantify the circulating cells.

הפרעה בקוטיקולה: שיטה לאיסוף המולימפה מזחלי דרוזופילה

Source: Reimels, T. A. and Pfleger, C. M. Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae. J. Vis. Exp. (2016).
The Drosophila ...

JoVE Encyclopedia of Experiments

Drosophila melanogaster (fruit fly)

Larva

Lipid Droplet Staining of Larval Drosophila Oenocytes: A Technique for Assessment of Lipid Droplet Accumulation Under Normal and Stressed Condition Using BODIPY-Based Dye

Source: Wei, C., et al., <a href="https://www.jove.com/v/60606">Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae.</a> J. Vis. Exp. (2019).
This video demonstrates lipid droplet staining in Drosophila larval oenocytes - hepatocyte-like cells - using the lipophilic fluorescent BODIPY based dye. These cells act as a model for studying lipid metabolism during development and in response to environmental stress such as starvation.

צביעת טיפות שומנים של Oenocytes Drosophila זחל: טכניקה להערכת הצטברות טיפות שומנים במצב רגיל ולחוץ באמצעות צבע מבוסס BODIPY

Source: Wei, C., et al., Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae. J. Vis. Exp. (2019).
This video demonstrates lipid ...

Biological Techniques

Staining Techniques

Cell staining

Larval Lymph Gland Dissection: Isolating the Drosophila Hemocyte-Producing Organ

Source: Reimels, T. A. and Pfleger, C. M. <a href="https://www.jove.com/video/54544/" target="_blank">Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae</a>. J. Vis. Exp. (2016).
This video describes the Drosophila lymph gland&#8212;the primary site of hematopoiesis in the fly larva&#8212;and shows an example protocol to dissect and mount the organ for immunohistochemistry and fluorescent imaging.

דיסקציה של בלוטת הלימפה בזחל: בידוד האיבר המייצר המוציטים של דרוזופילה

Source: Reimels, T. A. and Pfleger, C. M. Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae. J. Vis. Exp. (2016).
This video describes ...

שיטות לבדיקת בלוטת לימפה hemocytes ב

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אוכלוסיות תאי דם Assaying של

מבוא של צרעות טפיליות

ניתוח פונקציונלי של מעגל האכלת הזחל ב דרוזופילה