1. Animal model system Drosophila melanogaster
Why???
Graduate Institute of Biomedical Sciences,
Department of Biochemistry
Dr. Li-Mei Pai
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
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. Edward B.
Lewis
Christiane
Nüsslein-Volhard
Eric F.
Wieschaus

9. Body patterning of fly One cell to an organism
Figures\Chapter09\DevBio7e09070.jpg

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
Figures\Chapter09\DevBio7e09010.jpg

12. Gastrulation in Drosophila

13. Model of Drosophila Anterior-Posterior Pattern Formation
Maternal effect genes
Zygotic genes
Syncytial blastoderm
Figures\Chapter09\DevBio7e09081.jpg
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
Figures\Chapter09\DevBio7e09101.jpg

22. Figures\Chapter09\DevBio7e09102.jpg

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 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 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 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
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 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. 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
=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

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

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

53. The mechanism of localization of dorsal protein to the nucleus
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
Fig. 5-9

54. Nuclear gradient in dorsal protein
Fig. 5-14
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

55. Model for the subdivision of the dorso-ventral axis
into different regions by the gradient
in nuclear dorsal protein
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
Fig. 5-13

56. Dpp protein gradient
Cellularization---signal through transmembrane proteins
Dpp=BMP-4(TGF-b)
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