Methods
in developmental biology
Drosophila melanogaster
Drosophila melanogaster - common fruit fly (Diptera)
Life cycle
female male
• short generation time:
the shortest, seven days at 28 °C (30 °C: 11 days; 18 °C: 19 days; 12 °C: 50 days)
• lifespan 30 days at 29 °C
• high number of model animals: females produce about 500 eggs in 10 days
• embryonic development
• embryonic development (comm.)
• Eric Wieschaus talks: Patterning Development in the Embryo
3rd instar larva
48 hod
Advantages of Drosophila model
• one of the most studied organisms
• ease of breeding, space-saving, low cost
• well-known fate of individual cell populations
• genetically tractable
• at the molecular and cellular level, many developmental processes are similar to other species
• the basic model of eukaryotic genetics, embryogenesis, studies of maternal and homeotic genes, sex determination
• 4 pairs of chromosomes: female XX (1X:1A), male XY (1X:2A)
• haploid genome: 1.65 x 108 base pairs
• fertilization occurs in the oviduct, the eggs are laid as diploid
• superficial cleavage of eggs
• recombination occurs only in females
What do we need for fly handling?
• vials / bottles with caps
• fly food (water, yeast, syrup or sugar, agar, mashed potato powder or soy flour, ascorbic acid, fungicide Nipagin); formerly, crushed banana peel with yeast
• brushes and pad for manipulation
• anesthesia (C02, cold, ether, FlyNap® Anesthetic Kit)
• stereomicroscope
FlyBase - gene database and availability of fly lines
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FB2017_06: released Dec 31, 2017
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FlyExpress - gene expression maps
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FlyMove - developmental processes
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Stages
Processes
is]     Genetics Methods
Amnioserosa Dorsal vessel Epidermis Fat body Gonads Gut
Hemocytes Imaginal discs Lymph gland Malpighian tubules
Muscles Nervous system Ring gland Salivary glands Trachea
Organogenesis
Media Q
Within just one day the fertilized egg develops from a single cell into a complete and autonomous organism consisting of many different organs.
This section deals with the formation of the major organ systems from the cellular blastoderm stage onward.
At the blastoderm stage, the 6000 cells giving rise to all somatic tissues are located as a single cell wide epithelium at the surface of the embryo. Cell lineage analyses have shown, that the three germ layers - ectoderm, endoderm and mesoderm - are already determined at this stage. Thus, a so-called "anlagenplan" or fate map can be pojected onto the embryo.
• The ectoderm differentiates the nervous system, the epidermis, the fore- and hindgut, the salivary glands and malpighian tubules as well as the trachea and the ring gland.
• The two endoderm fractions represent the anlagen of the midgut.
• The mesoderm gives rise to all types of muscles, the fat body, the dorsal vessel, the
0 click to play
http://flymove.uni-muenster.de
Reporter lines
Haemocyte reporter line (hml-Gal4 UAS-RFP)     STAT pathway reporter line (STAT92E-GFP)
2XSTAT92E-GFP, yolk-GAL4/ + I UAS-hop/+; 2XSTAT92E-GFP. yolk-GAL4/ +
Bach et al. (2007) GFP reporters detect the activation of the Drosophila JAK/STAT pathway in vivo. https://doi.Org/10.1016/j.modgep.2006.08.003
UAS/Gal4 system for targeted gene expression
„driver line"      - specific promotor mediates spatial and temporal synthesis of Gal4
- Gal4 binds to DNA and activates transcription „responder line" - Upstream Activating Sequences bind Gal4 protein
- RNAi construct / GFP / copy of the target gene - its transcription is regulated by UAS which require Gal4 binding
UAS/Gal4 system for targeted gene expression - driver lines
• Actin-Gal4 (ubiquitous expression)
• hemolectin-Gal4 (embryonic/larval haemocytes; no lamellocyte expression observed)
• Hemese-Gal4 (haemocytes, embryonic/larval lymph gland)
• Lz-Gal4 (crystal cells)
• PPL-Gal4 (embryonic and larval fat body)
• NPl-Gal4(gut)
UAS/Gal4 system for targeted gene expression
• knock-down (UAS-RNAi, UAS-rpr), over-expression (UAS-gene sequence) or visualisation of target gene expression
• allows spacial and temporal control of gene expression (Gal4 drivers available both tissue and time-specific; eg. Bloomington)
• allows the maintenance of lethal mutations and mutations causing sterility (genotype is split into two fly lines and the harmful effect will not appear until both lines are crossed at 29 °C)
Critical steps of the use:
• the sequence of regulated gene must be known
• off-target effects
• rearing the flies at 29 °C
• non-complete knock-down (leaky expression)
The Nobel Prize in Physiology or Medicine 1995
The Nobel Assembly at the Karolínska Institute in Stockholm, Sweden, has awarded the Nobel Prize in Physiology or Medicine for 1995 to Edward B. Lewis, Christiane Nusslein-Volhard and Eric Wieschaus for their discoveries concerning "the genetic control of early embryonic development".
Photo from the Nobel Foundation      Photo from the Nobel Foundation      Photo from the Nobel Foundation
archive. archive. archive.
Edward B. Lewis Christiane Nusslein- Eric F. Wieschaus
Prize share: 1/3 Volhard Prize share: 1/3
Prize share:1/3
• the mechanisms of Wnt action have emerged from several systems: genetics in Drosophila melanogaster and Caenorhabditis elegans
• the Wnt homepage
materialstoday
Volume 14, Issue 5, May 2011, Pages 190-195
Review
Drosophila - a versatile model in biology 8^ medicine
Barbara H.Jennings^i El Show more
https://doi.org/10.1016/S1369-7021(ll)70113-4 Get rights and content
Under a Creative Commons license open access
Minireview
Posterior gut development in Drosophila: a model system for identifying genes controlling epithelial morphogenesis
Judith A Lengyel B & Xue Jun Liu
Cell Research 8,273-284(1998) | Cite this article
Open Access
J. Dev. Biol. 2017, 5(3), 9; https://doi.org/10.3390/jdb5030009
Editorial
Introduction: Drosophila—A Model System for Developmental Biology
by^ Nicholas S. Tolwinski1-2 B
1 Division of Science, Yale-NUS College, Singapore 138527, Singapore
2 Department of Biological Science, National University of Singapore, Singapore 138615, Singapore
Received: 19 September 2017 / Accepted: 20 September 2017 / Published: 20 September 2017
The Drosophila lymph gland as a developmental model of hematopoiesis
Seung-Hye Jung*, Cory J. Evans*, Christine Uemura and Utpal Banerjee*
Department of Molecular, Cell and Developmental Biology. Department of Biological Chemistry and Molecular Biology Institute. University of California, Los Angeles, CA 90095. USA
'These authors contributed equally to this work
'Author for correspondence (e-mail: baneriee@mbt.ucla.edu)
Accepted 29 March 2005
Development 132. 2521-2533
Published by The Company ot Biologists 2005
da:10.1242/dev.01837
Practical part
1) fly handling collection of virgins
visualisation of fat body using UAS/Gal4 system
2) preparing of D. melanogaster larvae for nanoinjection microscopy of reporter D. melanogaster lines
3) nanoinjection of D. melanogaster larvae and adults