Animal model system Drosophila melanogaster

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

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 evolutionPax6 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 Wild type Ligand (signal) Receptor (torpedo) 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 Imaginal disc Easily to be cultured , large population

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

9. Body patterning of fly One cell to an organism

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

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 Egg shell Fig. 5-10 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

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. A/P Determination during oogenesis 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.

17. Axis Determination during oogenesis • Gurken—TGF-a • Torpedo--EGFR • 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. Approach I: transplantation The bicoid gene is necessary for the establishment of the anterior structure Bicoid--fertilized—translated Protein diffuses and forms morphogen gradient No head and thoracic Prick at the anterior of normal egg Partial rescued

24. Approach II: expression patternThe 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 copiesMaternal bicoid protein controls zygotic hunchback expression Dosages of maternal bicoid genes Bicoid = homeodomain transcription factor

28. Approach V transcriptional regulationP-element mediated transformation-hunchback expression

29. Thresholds of Transcription factorkrÜppel gene activity is specified by Hunchback protein kruppel is the 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 networkThe 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 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 The expression of the engrailed gene Anterior margin of each parasegment

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 expressionholoprosencephaly,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. Bithorax mutant –PS 4 default state +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-a • Torpedo--EGFR • posterior • dorsal http://www.youtube.com/watch?v =GntFBUa6nvs Fig. 5-12

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

49. Torpedo--EGFR

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