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Flies as a model for the study of human disease

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Presentation on theme: "Flies as a model for the study of human disease"— Presentation transcript:

1 Flies as a model for the study of human disease
Rapid construction of transgenic models of human disease Well established easy systems to drive knockdown/knockout or over expression of gene expression in tissue or temporal specific patterns Rapid forward genetics – isolate mutants through transposons or chemical mutagenesis Rapid determination of the molecular basis of disease mechanisms Able to rapidly identify modifier/bypass gene pathways via genetic screens for enhancers or suppressors of phenotypes Easy to culture cell lines – very-easy to dsRNA treat genes of interest

2 The fly GAL4/UAS binary transgenic expression system
OFF Tissue specific promoter GAL4 UAS Transgene GAL4 is a transcriptional activator protein from yeast The upstream activating sequence is the GAL4 target GAL4/UAS from yeast ON Tissue specific promoter GAL4 UAS Transgene The progeny of this mating will express the transgene in cells also expressing GAL4

3 The life cycle of a fly Adult Pupa White (early) pupa L3 L2
Starting at bottom left Flies lay ~2eggs/hour They hatch after 24 hours into a juvenile form (Larva) that molts three times (L1-L2-L3) Larva primarily feed and grow. Note that adult structures are formed within the larva but remain quiescent until puparation Maturation from the juvenile form to adult is induced by hormonal changes - the larvae forms hard outer shell and the outer aspects of juvenile form are degraded and replaced by the adult tissues. The CNS and other critical organs are retained through puparation. The adult fly emerges (elcosion) from the pupal case. L2 L1 Larva (juvenile) 24 hrs Egg

4 Drosophila has 4 chromosomes
H. sapiens chromosomes

5 Embryonic Brain Development
The embryonic brain development (Stage 11) – pCenBr – Central Brain Nb - neuroblasts pVisSys – Visual System – underdeveloped in embryos as larvae have no eyes gmc – ganglion mother cells mp – midline glial cells pVenNC – Ventral Nerve Cord OL – optic lobe cn – longitudinal connective neurons co – commissures (2 per segment) Hartenstein - Atlas of Drosophila Development (1993) Cold Spring Harbour Laboratory Press

6 Larval/Pupal Brain Development
OL – optic lobe OOA – outer optic alnage CenBR – Central Brain VenNC – Ventral Nerve Cord SubGgl - subesophageal ganglion (three fused gaglia) – multiple neuronal types ThAGgl - horacico-abdominal ganglion – regulation of locomotors behaviour of legs/wings – analogous to spine Hartenstein - Atlas of Drosophila Development (1993) Cold Spring Harbour Laboratory Press

7 The fly fat body is analogues to adipose tissue, the liver and the haematopoietic system in mammals.
The fly liver, fat and bone marrow analogues are one tissue. Hotamisilgil (2006) Inflammation and metabolic disorders Nature 444,

8 Drosophila oenocytes are analogous to mammalian hepatocytes
Hormone names are shown in red, tissue names in blue. DILP,Drosophila insulin-like peptides; TAG: triacylglycerol. Leopold & Perrimon (2007) Drosophila and the genetics of the internal milieu Nature 450,

9 Testing drug candidates in flies
Throughput Routes of drug administration. For larva (top), drug can be directly injected or drug can be mixed with media. Media can be either solid or liquid with 2% yeast paste to encourage feeding behavior. Adults can have drug delivered as an aerosol or gas, as a mixture with food substrate, as a direct application to exposed nerve cord, or as an injection. Drug administration through feeding generally has the highest throughput. For our screens – we will use of mildly affected larvae - or feeding/injection into the ovaries of adult females to test effect(s) on eggs. Pandley and Nichols (2011) Human Disease Models in Drosophila melanogaster and the Role of the Fly in Therapeutic Drug Discovery Pharmacological Reviews 63(2)

10 ‘Humanized” fly Pex1 mutations

11 ‘Humanized” fly Pex1 mutations

12

13 A ‘visible’ screen for peroxisome function in Drosophila eyes
ey – eyeless:GAL4 GMR– Glass Multiple Reporter:GAL4 For both Pex3, Pex7 and Pex 16 we can we can see that when peroxisome function is inhibited specifically in the eye via dsRNA knockdown we see a change in eye colour = Beard and Holtzman showed that the ‘rosy’ eye colour is linked to differences in peroxisome number. Rosy mutation is a deletion in the structural gene for xanthine dehydrogenase. Thus, the rosy-506 mutation appears to affect peroxisomes and may mimic aspects of the defects of peroxisomes in some human metabolic disorders.

14 Drosophila Pex1 is expressed throughout development
*RNAseq

15 Drosophila Pex1 is expressed in multiple tissues
*microarray Malpigial tubules – analogous to some but not all functions of human kidney

16 Drosophila Pex3 is expressed in multiple tissues
*microarray

17 Drosophila Pex7 is expressed highly in the CNS
*microarray Note Pex7 is only expressed significantly in the CNS

18 Drosophila Pex1

19 Genome wide analysis of Pex1 loss

20 Genome wide analysis of Pex1 loss

21 Genome wide analysis of Pex1 loss

22 Genome wide analysis of Pex1 loss

23 Loss of Pex1 in flies causes larval lethality

24 Loss of Pex1 in flies causes a poor growth phenotype

25 Pex1 mutations do not affect fly musculature

26 Loss of Pex1 in flies causes a poor growth phenotype

27 Loss of Pex1 in flies causes severe effects on the Drosophila nervous system

28 Loss of Pex1 in flies causes severe effects on the Drosophila nervous system

29 Loss of Pex1 in flies causes severe effects on the Drosophila nervous system

30 High Throughput screening
dsRNA library covering 96% of the Drosophila genome High throughput screening

31 Studying Peroxisomes in cultured fly cells
The S2 cell line was derived from a primary culture of late stage (20-24 hours old) Drosophila melanogaster embryos. Many features of the S2 cell line suggest that it is derived from a macrophage-like lineage. These are a stable cell line where peroxisomes are marked by GFP-SKL- gift from Ron Vale laboratory

32 Acknowledgements McGill Univeristy U of Alberta Dr. Nancy Braverman
Simmonds Laboratory Jing Li Julie Haskins Alana Pay Rachubinski Laboratory Jenny Chang Fred Mast Robert Tower Rick Porier Dr. Sarah Hughes McGill Univeristy Dr. Nancy Braverman


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