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Animal model system Drosophila melanogaster Graduate Institute of Biomedical Sciences, Department of Biochemistry Dr. Li-Mei Pai Why??? 醫學一 8F, 5520

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Presentation on theme: "Animal model system Drosophila melanogaster Graduate Institute of Biomedical Sciences, Department of Biochemistry Dr. Li-Mei Pai Why??? 醫學一 8F, 5520"— Presentation transcript:

1 Animal model system Drosophila melanogaster Graduate Institute of Biomedical Sciences, Department of Biochemistry Dr. Li-Mei Pai Why??? 醫學一 8F, 5520

2 My exploration in Science 東吳大學 微生物 (Bachalor) 陽明大學 微免所 (Master-EBV) 美國 北卡州立大學 教堂山分校 ( The University North Carolina, Chapel Hill—Ph.D) Thesis: The function of Drosophila armadillo gene (Development, 1997) 美國 普林斯敦 大學 (Princeton University—Postdoctoral fellow) Study: Identify Cbl oncogene in Drosophila body patterning (Cell, 2000)

3 Functional homologous genes during evolution Pax6 and Eyeless Homologous genes initiate the development program for the same organ in animals separate by 500 million years of evolution

4 Genes & Development Mutants in the EGFR signaling pathway Little gene redundancy Receptor (torpedo) Wild type Ligand (signal) GAP (negative regulator)

5 Fewer genes 4 pairs of chromosomes

6 Functions of 13,600 genes??? Development of the Drosophila body plan Axis determination Signaling pathway Transcriptional and translational regulationfunctions

7 Life cycle of Drosophila (very short ) 4 stages: embryo, larva, pupa, adult Easily to be cultured, large population Imaginal disc

8 Edward B. Lewis Christiane Nüsslein-Volhard Eric F. Wieschaus

9 Body patterning of fly One cell to an organism

10 Genetic screening strategy for identifying developmental mutants More than 100 genes!! How to know who are they?? tomorrow b:balancer DTS; dominant Temp. sensitive

11 Superficial Cleavage in a Drosophila early Embryo Syncytial blastoderm

12 Gastrulation in Drosophila

13 Model of Drosophila Anterior-Posterior Pattern Formation Maternal effect genes Zygotic genes Syncytial blastoderm Cellular blastoderm

14 Egg development in Drosophila each egg chamber: 3 types of cells Oocyte with nucleus (germinal vesicle-GV) Connected to 15 nurse cells }---germ-line Surrounded by a monolayer of about 1000 somatic follicle cells Egg shell Fig. 5-10

15 Signals from older to younger egg chambers Red arrow: Delta-Notch induces anterior polar follicle cells JAK-STAT: form the stalk cells Yellow arrow: signals induce E-cadherins expression

16 The oocyte move towards one end in contact with follicle cells Both the oocyte and the posterior follicle cells express high levels of the E-cadherin If E-cadherin is removed, the oocyte is randomly positioned. Then the oocyte induces surrounding follicle cell to adopt posterior fate. A/P Determination during oogenesis

17 Axis Determination during oogenesis Gurken—TGF-  Torpedo--EGFR 1.posterior Fig. 5-12

18 mRNA localization in the oocyte Dynein-gurken and bicoid to the plus end Kinesin—oskar to the minus end

19 The sequential expression of different sets of genes establishes the body plan along the anterior- posterior axis Localized mRNA and Proteins Translated after fertilization— Temporal sequence

20 The effects of mutations in the maternal gene system Three classes Anterior Posterior terminal head and thoracic abdominal acron and telson

21 Independent Genetic Pathways Interact to Form the Anterior-Posterior Axis


23 Bicoid--fertilized—translated Protein diffuses and forms morphogen gradient No head and thoracic Prick at the anterior of normal egg Partial rescued Approach I: transplantation The bicoid gene is necessary for the establishment of the anterior structure

24 Approach II: expression pattern The distribution of the maternal mRNA and protein of bicoid Short Half life Transcription factor--- Activates zygotic gene In situ RNA hybridization Immunostaining Antibody interaction

25 Approach III: relationship between genes Posterior determination 9 maternal genes Abnormal abdominal Development Oskar localizes nanos mRNA Nanos suppresses the translation of the maternal mRNA of Hunchback(hb)

26 The expression of Gap genes First zygotic genes—transcription factors Mutant –large section of the body is missing Blastoderm—proteins diffuse away but with short half life

27 Approach IV: the effects of gene copies Maternal bicoid protein controls zygotic hunchback expression Dosages of maternal bicoid genes Bicoid = homeodomain transcription factor

28 Approach V transcriptional regulation P-element mediated transformation -hunchback expression

29 Thresholds of Transcription factor krÜppel gene activity is specified by Hunchback protein kruppel is t he target genes of hunchback Increase dose of hunchback – kruppel shift posteriorly

30 The striped patterns of activity of pair-rule genes Pair-rule genes in 14 segments Even-skipped—odd-number Fusi tarazu—even number Syncytium just before cellularization Each stripe is specified independently

31 Transcription network The specification of the second even-skipped (eve) stripe by gap gene proteins Bicoid and Hb activate eve Kruppel and Giant repress eve

32 Sites of action of activating and repressing transcription factors

33 Segment polarity A/P axis within one segment Ventral epidermis of the abdomen—ventral denticle belts (anterior) Mutation—alter the denticle pattern Wingless=Wnt hedgehog

34 The cuticle of each segment in the abdomen of the adult Drosophila Different bristles, pigmentation, and gene expression en- clone—anterior type cuticle

35 Segment polarity genes and compartment The expression of the engrailed gene Anterior margin of each parasegment Mutations upset the A/P polarity of the segment They are activated in response to pair-rule genes Engrailed (en) —cell lineage boundary, defines a compartment En: homeodomain transcription factor

36 Interactions between hedgehog, wingless, and engrailed hh turn on wg expression, wg maintain en expression

37 The hedgehog signaling pathway Without signal—Ci is processed as a repressor into nucleus With signal---full length Ci acts as an activator in the nucleus

38 SHH mutation-50% reduction in gene expression holoprosencephaly,or failure of the midface and forebrain to develop (cleft lip and palate, hypotelorism) Signaling pathways are conserved-receptor on the target cells, intracellular effectors, changes in the activity of the target transcription factor

39 Malformation: Polydactyly and syndactyly abnormalities in one or more genetic programs Greig cephalopolysyndactyly (GCPS): loss of function mutation in GLI3 (Ci) —transcription factor

40 The wingless signaling pathway More than 50% Colon cancr with Mutation in APC C-myc target gene

41 Metamorphosis

42 Homeotic selector genes Each segment unique identity—master regulator genes Homeotic selector genes—control other genes-required throughout development Vertebrate Hox gene complex

43 Homeotic transformation of the wing and haltere Homeotic genes—mutated into homeosis transformation As positional identity specifiers Bithorax-haltere into wing

44 The spatial pattern of expression of genes of the bithorax complex Bithorax—Ultrabithorax –5-12 Abdominal-A—7-13 Abdominal-B—10-13 Bithorax mutant –PS 4 default state

45 +Ubx—5,6 +Abd-A—7,8,9 +Abd-B—10 Combinatorial manner

46 Mutation in HoxD13—synpolydactyly Extra digits & interphalangeal webbing (hetero) Similar but more severe & bony malformation of hands, wrists (Homo)

47 Axis Determination during oogenesis Gurken—TGF-  Torpedo--EGFR 1.posterior 2.dorsal Fig. 5-12 =GntFBUa6nvs

48 The EGFR signal establishes the D-V axial pattern of the egg chamber Fig. 5-11 Gurken—TGF-  green) Actin-cell outline (red) Blue-dorsal anterior Follicle cells

49 Torpedo--EGFR

50 The Key determinant in D/V polarity is pipe mRNA in follicle cells

51 windbeutel—ER protein pipe—heparansulfate 2-o-sulfotransferase (Golgi) nudel—serine protease The activation of Toll

52 Toll protein activation results in a gradient of intranuclear dorsal protein Spatzle is processed in the perivitelline space after fertilization Fig. 5-8

53 1.Toll mutant – dorsalized (no ventral structure) 2. Transfer wt cytoplasm into Toll mutant specify a new dorsal-ventral axis (injection site =ventral side) Without Toll activation Dorsal + cactus Toll activation – tube (adaptor) and pelle (kinase) Phosphorylate cactus and promote its degradation B cell gene expression Dorsal=NF-kB Cactus=I-kB The mechanism of localization of dorsal protein to the nucleus Fig. 5-9

54 Dorsalized embryo— Dorsal protein is not in nuclei Dpp is everywhere Twist and snail are not expressed Threshold effect—integrating Function of regulatory binding sites Regulatory element =developmental switches Nuclear gradient in dorsal protein Fig. 5-14

55 Zygotic genes pattern the early embryo Dorsal protein activates twist and snail represses dpp, zen, tolloid Rhomboid----neuroectoderm Repressed by snail (not most ventral) Binding sites for dorsal protein in their regulatory regions Model for the subdivision of the dorso-ventral axis into different regions by the gradient in nuclear dorsal protein Fig. 5-13

56 Dpp protein gradient Cellularization---signal through transmembrane proteins Dpp=BMP-4(TGF-  ) Dpp protein levels high, increase dorsal cells short of gastrulation (sog) prevent the dpp spreading into neuroectoderm Sog is degraded by Tolloid (most dorsal)

57 References: 1. Principles of Development 2nd edition, by Lewis Wolpert (P48-52) 2. The genetics of axis specification in Drosophila The Chapter 9 of Developmental Biology by Scott Gilbert, 9th edition

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