1. http://flymove.uni-muenster.de/Processes/Segmentation/SegmentGes.html

2. WT wg

4. A>P <A loss of P <A A> P P Mirror image duplication

5. WT en

6. WTen anterior posterior

7. http://flymove.uni-muenster.de/Processes/Segmentation/SegmentGes.html

8. CHRISTIANE NUSSLEIN-VOLHARD Max Planck Institute Tuebingen, Germany

9. Saturation mutagenesis: -identify mutations in all possible genes involved in embryo patterning -saturation reached at ~5 mutations per gene

10. Saturation mutagenesis: -saturation reached at ~5 mutations per gene tossing marbles into bins How many bins are there? -identify mutations in all possible genes involved in embryo patterning

11. Saturation mutagenesis: -saturation reached at ~5 mutations per gene 15 marbles tossed into bins 5 5 5 Probability of missing fourth bin in 15 trys: -identify mutations in all possible genes involved in embryo patterning

12. Saturation mutagenesis: -saturation reached at ~5 mutations per gene 15 marbles tossed into bins 5 5 5 0.75 15 = 0.013 Probability of missing fourth bin in 15 trys: -identify mutations in all possible genes involved in embryo patterning

13. Saturation mutagenesis: -saturation reached at ~5 mutations per gene 15 marbles tossed into bins 5 5 5 0.75 15 = 0.013 Probability of missing fourth bin in 15 trys: ~99% probability there are only 3 bins -identify mutations in all possible genes involved in embryo patterning

14. Genes that control pattern and polarity in the embryo: Maternal genes: 1. anterior-posterior -bicoid -nanos 2. terminal -torso 3. dorsal ventral anterior posterior dorsal ventral Zygotic genes: 1. Gap genes 2. Pair-rule genes 3. Segment polarity genes 4. Homeotic genes early late

15. Triple mutant: -no information nanos, torso double: -only bicoid bicoid, torso double: -only nanos bicoid, nanos double: -only torso

16. Maternal genes: Bicoid Hunchback Gap genes: Hunchback Kruppel Knirps Giant Pair-rule genes: Even-skipped Ftz Runt Prd Odd-skipped Segment polarity genes: Engrailed Wingless Decapentaplegic Hedgehog Homeotic genes: Ubx abdA abdB Antp

17. WTknirps

18. Knirps phenotype expression

19. Kruppel Mirror image duplication phenotype expression

20. Maternal genes: Bicoid Hunchback Gap genes: Hunchback Kruppel Knirps Giant Pair-rule genes: Even-skipped Ftz Runt Prd Odd-skipped Segment polarity genes: Engrailed Wingless Decapentaplegic Hedgehog Homeotic genes: Ubx abdA abdB Antp

21. Pair rule: Even-skipped phenotype expression

22. Hairy Runt Eve Ftz

23. Segment polarity: patched

25. WT wg

26. Maternal genes: Bicoid Hunchback Gap genes: Hunchback Kruppel Knirps Giant Pair-rule genes: Even-skipped Ftz Runt Prd Odd-skipped Segment polarity genes: Engrailed Wingless Decapentaplegic Hedgehog Homeotic genes: Ubx abdA abdB Antp

29. Lecture 3…

32. WT en

33. WT wg

36. http://www.ucalgary.ca/UofC/eduweb/virtualembryo/D_m_segment_I.html

37. knirps kruppel giant Even-skipped Gap genes Pair-rule stripes

40. bicoid hunchback knirps

41. Knirps phenotype expression

43. Ma et al. (1996) Development 122 (4): 1195. BICOID bindings sites in the Hunchback and Knirps enhancers The EMBO Journal (1998) 17, 5998–6009 Hunchback Knirps

44. The EMBO Journal (1998) 17, 5998–6009

45. Cell. 2007 Jul 13;130(1):141-52. Cell. 2007 Jul 13;130(1):153-64.

47. Is cooperativity sufficient? Spatial Bistability Generates hunchback Expression Sharpness in the Drosophila Embryo Francisco J. P. Lopes1,2,3*, Fernando M. C. Vieira3,4, David M. Holloway5,6,7, Paulo M. Bisch3, Alexander V. Spirov1,2 PLOS Computational Biology (2008) 4:e1000184

49. BCD Hb n=~5 Positive feedback loop + Cooperative binding

50. Bi-stable dynamics

51. BCD Hb n=~5 Positive feedback loop + Cooperative binding

52. BCD Hb n=~5 Cooperative binding No feedback

53. No feedback – no bi-stability

54. lowering Hill co-efficient shifts curve, but still bi-stable

56. http://www.ucalgary.ca/UofC/eduweb/virtualembryo/D_m_segment_I.html

57. Giant Kruppel Giant

58. Wild Type bicoid Giant expression

59. Kruppel Mirror image duplication phenotype expression

60. Knirps phenotype expression

62. Pair rule: Even-skipped phenotype expression

63. knirps kruppel giant Even-skipped Concentration dependent effects of Hunchback Bicoid Fishhook

65. WT eve expression Stripe 2- enhancer lacZ Stripe 3+7 enhancer lacZ

66. WT kni hb kni tor kni hb tor Gap gene regulation of stripe 3

67. Stage 14 Later cellularized Knirps defines boundaries of stripe 3 and 7

82. Bicoid binding site deleted Expression partially restored by compensating removal of Giant repressor site

83. Deletion of GIANT binding sites expands band Even-skipped expression

84. Lecture 2 -segment polarity -wing polarity -evolution of development

86. WT wg

87. WTen anterior posterior

89. Segment Parasegment A P A P A P

90. EvenOddEven Parasegments Segments denticals wg en runt prd eve ftz prd runt ftz prd runt

91. Hairy Runt Eve Ftz

92. EvenOddEven Parasegments runt eve hairy hunchback giant kruppel bicoid

96. EvenOddEven Parasegments Segments denticals wg en runt prd eve ftz prd runt ftz prd runt

97. Hedge hog

98. Segment polarity: patched

99. En mRNA Hh protein Hh is a short range signal

100. wg en pppp ptc Cells on the en side secrete hh peptide, but lack the hh receptor encoded by ptc In the absence of the hh peptide wg is repressed by the ptc signalling pathway hh wg hh wg en ci

102. Armadillo (beta-catenin) frizzled wg TCF dsh

104. gene phenotypevertebrate ortholog Wingless (Wnt1) segment polarity, many others Wnt1 DWnt2Pigment cells gonads; Kozopas 1998Wnt7 adult muscle Kozopas 2002 Trachea (with wg) Llimargas 2001 DWnt3/5 Axon Guidance (through Derailed)Wnt5 Yoshikawa 2003 DWnt4Cell Movement in ovary Cohen 2002Wnt9 Dorsoventral specificity of retinal projections (Sato 2006) DWnt6 Wnt6 DWnt8Antagonist Dorsal, no ortholog immunity phenotype (Gordon et al, 2005; Ganguly et al, 2005) DWnt10 Wnt10

105. Gene Phenotype of Knockouts or other functions 19 mammalian Wnts Wnt1midbrain, cerebellu; neural crest derivatives; hymocyte number Wnt2placental defects Monkley, 1996 Wnt2b/13retinal cell differentiation Kubo, 2003, Kubo, 2005 Wnt3early gastrulation defect; Axis formation; Hair growth; medial-lateral retinotectal topography; hippocampal neurogenesis Wnt3a vestigial tail; neural crest; hippocampus;Segmentation oscillation clock; left right asymmetry Wnt4kidney defects; sex determination; side-branching in mammary gland; number of thymocytes migration of steroidogenic adrenal precursors into the gonad Jeays-Ward 2003 Anterior-posterior guidance of commissural axons. Wnt5a truncated limbs, truncated AP axis, reduced number proliferating cells Yamaguchi 1999 Distal lung morphogenesi; chondrocyte differentiation, longitudinal skeletal outgrowth; Inhibits B cell proliferation and functions as a tumor suppressor Defects in posterior growth of the female reproductuve tract Wnt5b Wnt6 Wnt7alimb polarity; uterine patterning during the development of the mouse female reproductive tract Delayed maturation synapses in Cerebellum Wnt7bPlacental developmen; lung hypoplasia; macrophage-induced programmed cell death Wnt8a Wnt8b Wnt9aJoint integrity Wnt9bmesenchymal to epithelial transitions Wnt10a Wnt10b decreased trabecular bone; myogenic and Adipogenic program; overexpression inhibits adipogenesis Wnt11Ureteric branching defects; cardiogenesis Wnt16

107. WTen anterior posterior

108. ptc en

109. dpp en ptc Cells on the en side secrete hh peptide, but lack the hh receptor encoded by ptc In the absence of the hh peptide dpp is repressed by the ptc signalling pathway hh dpp hh dpp en

110. ptc en

112. MAD signaling factor is activated by the dpp receptor: phosphorylation state of MAD forms a signaling gradient

113. Conservation of Hedgehog

118. haltere

120. ANTp loss of function transforms leg to antenna-like appendage

121. Antennapedia Wild type Antp c.a. 1949

124. Ubx mutant ? ?

125. fly Dragon fly crustacean Ubx abd A Evolution of development

127. ~550 MYA

131. Segment polarity: patched

132. wg en runt prd eve ftz ptc

133. En en-  En en-  En en-  het DNA replication Mitotic Cross-over Mutant daughter cell

134. Creating genetic mosaic flies by mitotic recombination

135. WTen anterior posterior

136. Hunchback is sensitive to ~10% changes in BICOID protein concentration

137. 1. The gradient is approximately (but not necessarily exactly) an exponential decay in intranuclear (and cytoplasmic) concentration that is established rapidly (less than 90 min). 2. Bcd diffuses relatively slowly (D = 0.3 mm2/s) in the cortical cytoplasm containing the nuclei. 3. The Bcd gradient is stable over nuclear cycles 10– 14, when the number of nuclei is growing by a factor of two with each division and Bcd is concentrated and released from nuclei in a dynamic process. In particular, the initial postinterphase concentration in nuclei in successive cycles is constant to at least 10%. 4. Bcd is not simply trapped in nuclei; rather, it is in dynamic equilibrium between influx and efflux with the cytoplasm and, possibly, intranuclear degradation. 5. The spatial shape of the Bcd gradient (the length constant for an exponential decay) scales with embryo length over a factor of five range in lengths in different dipteran species (Gregor et al., 2005).