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Abstract

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Protocol

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Materials

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Developmental Biology

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Published: November 28th, 2016

DOI:

10.3791/54544

1The Graduate School of Biomedical Sciences, The Icahn School of Medicine at Mount Sinai, 2Department of Oncological Sciences, The Icahn School of Medicine at Mount Sinai

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.

Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

The complex mechanisms regulating the transcription factors and signaling pathways that coordinate the development of the hematopoietic system and that malfunction in hematological diseases remain poorly understood. These transcription factors and signaling pathways, as well as their regulation, are highly conserved between Drosophila and mammalian hematopoiesis1-5. Thus the Drosophila hematopoietic system represents an excellent genetic model to define the molecular mechanisms controlling hematopoiesis and underlying hematological diseases.

Similar to mammals, Drosophila generate blood cells, called he....

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1. Circulating Hemocyte Concentration

  1. To obtain larvae of roughly the same developmental stage for this assay, restrict egg collection by allowing females to lay eggs for a fixed time period of 2 - 6 hr.
  2. Collect larvae in dissecting dish wells filled with 1x phosphate buffered saline (PBS, Table 1).
  3. For each larva, place 10 µl 1x PBS in a microcentrifuge tube on ice and 10 µl 1x PBS on a clean dissecting pad. Place the dissecting pad on an illuminated stereomicro.......

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Circulating Hemocyte Concentration

Hemocyte numbers increase throughout larval development35. To illustrate that this method detects differences in hemocyte numbers and concentration, regardless of the biological cause, we measured hemocyte concentrations of delayed and non-delayed larvae. Loss of prothoracicotropic hormone (ptth) by genetic ablation of ptth-producing neurons (ptth>grim) produces a delay in larval de.......

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Upon genetic or environmental alteration, the four methods described here can be used individually or in conjunction to analyze distinct processes during hematopoiesis such as signaling, survival, proliferation, and differentiation. Drosophila hematopoiesis is a dynamic process; the number of hemocytes per animal increases35 and the structure and gene expression of the lymph gland changes32 during development. Prior to performing these assays, therefore, it is critical to restrict egg colle.......

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

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

  1. Evans, C. J., Hartenstein, V., Banerjee, U. Thicker than blood: conserved mechanisms in Drosophila and vertebrate hematopoiesis. Dev Cell. 5 (5), 673-690 (2003).
  2. Crozatier, M., Meister, M. Drosophila haematopoiesis. Cell Microbiol. 9 (5), 1117-1126 (2007).
  3. Crozatier, M., Vincent, A. Drosophila: a model for studying genetic and molecular aspects of haematopoiesis and associated leukaemias. Dis Model Mech. 4 (4), 439-445 (2011).
  4. Gold, K. S., Bruckner, K. Drosophila as a model for the two myeloid blood cell systems in vertebrates. Exp Hematol. 42 (8), 717-727 (2014).
  5. Hartenstein, V. Blood cells and blood cell development in the animal kingdom. Annu Rev Cell Dev Biol. 22, 677-712 (2006).
  6. Ghosh, S., Singh, A., Mandal, S., Mandal, L. Active hematopoietic hubs in Drosophila adults generate hemocytes and contribute to immune response. Dev Cell. 33 (4), 478-488 (2015).
  7. Leitao, A. B., Sucena, E. Drosophila sessile hemocyte clusters are true hematopoietic tissues that regulate larval blood cell differentiation. Elife. 4, (2015).
  8. Makhijani, K., Alexander, B., Tanaka, T., Rulifson, E., Bruckner, K. The peripheral nervous system supports blood cell homing and survival in the Drosophila larva. Development. 138 (24), 5379-5391 (2011).
  9. Crozatier, M., Ubeda, J. M., Vincent, A., Meister, M. Cellular immune response to parasitization in Drosophila requires the EBF orthologue collier. PLoS Biol. 2 (8), 196 (2004).
  10. Markus, R., et al. Sessile hemocytes as a hematopoietic compartment in Drosophila melanogaster. Proc Natl Acad Sci U S A. 106 (12), 4805-4809 (2009).
  11. Minakhina, S., Steward, R. Melanotic mutants in Drosophila: pathways and phenotypes. Genetics. 174 (1), 253-263 (2006).
  12. Bina, S., Wright, V. M., Fisher, K. H., Milo, M., Zeidler, M. P. Transcriptional targets of Drosophila JAK/STAT pathway signalling as effectors of haematopoietic tumour formation. EMBO Rep. 11 (3), 201-207 (2010).
  13. Avet-Rochex, A., et al. An in vivo RNA interference screen identifies gene networks controlling Drosophila melanogaster blood cell homeostasis. BMC Dev Biol. 10, 65 (2010).
  14. Rodriguez, A., et al. Identification of immune system and response genes, and novel mutations causing melanotic tumor formation in Drosophila melanogaster. Genetics. 143 (2), 929-940 (1996).
  15. Mandal, L., Banerjee, U., Hartenstein, V. Evidence for a fruit fly hemangioblast and similarities between lymph-gland hematopoiesis in fruit fly and mammal aorta-gonadal-mesonephros mesoderm. Nat Genet. 36 (9), 1019-1023 (2004).
  16. Grigorian, M., Mandal, L., Hakimi, M., Ortiz, I., Hartenstein, V. The convergence of Notch and MAPK signaling specifies the blood progenitor fate in the Drosophila mesoderm. Dev Biol. 353 (1), 105-118 (2011).
  17. Lebestky, T., Jung, S. H., Banerjee, U. A Serrate-expressing signaling center controls Drosophila hematopoiesis. Genes Dev. 17 (3), 348-353 (2003).
  18. Krzemien, J., et al. Control of blood cell homeostasis in Drosophila larvae by the posterior signalling centre. Nature. 446 (7133), 325-328 (2007).
  19. Mandal, L., Martinez-Agosto, J. A., Evans, C. J., Hartenstein, V., Banerjee, U. A Hedgehog- and Antennapedia-dependent niche maintains Drosophila haematopoietic precursors. Nature. 446 (7133), 320-324 (2007).
  20. Benmimoun, B., Polesello, C., Haenlin, M., Waltzer, L. The EBF transcription factor Collier directly promotes Drosophila blood cell progenitor maintenance independently of the niche. Proc Natl Acad Sci U S A. 112 (29), 9052-9057 (2015).
  21. Sinenko, S. A., Mandal, L., Martinez-Agosto, J. A., Banerjee, U. Dual role of wingless signaling in stem-like hematopoietic precursor maintenance in Drosophila. Dev Cell. 16 (5), 756-763 (2009).
  22. Pennetier, D., et al. Size control of the Drosophila hematopoietic niche by bone morphogenetic protein signaling reveals parallels with mammals. Proc Natl Acad Sci U S A. 109 (9), 3389-3394 (2012).
  23. Dragojlovic-Munther, M., Martinez-Agosto, J. A. Extracellular matrix-modulated Heartless signaling in Drosophila blood progenitors regulates their differentiation via a Ras/ETS/FOG pathway and target of rapamycin function. Dev Biol. 384 (2), 313-330 (2013).
  24. Ferguson, G. B., Martinez-Agosto, J. A. Yorkie and Scalloped signaling regulates Notch-dependent lineage specification during Drosophila hematopoiesis. Curr Biol. 24 (22), 2665-2672 (2014).
  25. Milton, C. C., et al. The Hippo pathway regulates hematopoiesis in Drosophila melanogaster. Curr Biol. 24 (22), 2673-2680 (2014).
  26. Evans, C. J., Liu, T., Banerjee, U. Drosophila hematopoiesis: Markers and methods for molecular genetic analysis. Methods. 68 (1), 242-251 (2014).
  27. Neyen, C., Bretscher, A. J., Binggeli, O., Lemaitre, B. Methods to study Drosophila immunity. Methods. 68 (1), 116-128 (2014).
  28. Small, C., Paddibhatla, I., Rajwani, R., Govind, S. An introduction to parasitic wasps of Drosophila and the antiparasite immune response. J Vis Exp. (63), e3347 (2012).
  29. Petraki, S., Alexander, B., Bruckner, K. Assaying Blood Cell Populations of the Drosophila melanogaster Larva. J Vis Exp. (105), (2015).
  30. Rizki, M. T. M., Rizki, R. M. Functional significance of the crystal cells in the larva of Drosophila mekmogaster. Journal of Biophysical and Biochemical Cytology. 5, 235-240 (1959).
  31. Kurucz, E., et al. Definition of Drosophila hemocyte subsets by cell-type specific antigens. Acta Biol Hung. 58, 95-111 (2007).
  32. Jung, S. H., Evans, C. J., Uemura, C., Banerjee, U. The Drosophila lymph gland as a developmental model of hematopoiesis. Development. 132 (11), 2521-2533 (2005).
  33. Krzemien, J., Crozatier, M., Vincent, A. Ontogeny of the Drosophila larval hematopoietic organ, hemocyte homeostasis and the dedicated cellular immune response to parasitism. Int J Dev Biol. 54 (6-7), 1117-1125 (2010).
  34. Rizki, T. M., Rizki, R. M. Properties of the Larval Hemocytes of Drosophila-Melanogaster. Experientia. 36 (10), 1223-1226 (1980).
  35. Lanot, R., Zachary, D., Holder, F., Meister, M. Postembryonic hematopoiesis in Drosophila. Dev Biol. 230 (2), 243-257 (2001).
  36. McBrayer, Z., et al. Prothoracicotropic hormone regulates developmental timing and body size in Drosophila. Dev Cell. 13 (6), 857-871 (2007).
  37. Reimels, T. A., Pfleger, C. M. Drosophila Rabex-5 restricts Notch activity in hematopoietic cells and maintains hematopoietic homeostasis. J Cell Sci. 128 (24), 4512-4525 (2015).

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