Wang2012_2.pdf

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3. for their discoveries concerning the genetic control of early embryonic development
4. Nobel committee awarded John Gurdon and Shinya Yamanaka the prize in physiology or medicine for discovering that ordinary cells of the body can be reprogrammed into stem cells, a discovery which may lead to new treatments. Nobel prize
5. The agenda 1) Briefly summarize the paper 2) Who cares [about epithelial folding]? 3) A closer look at the paper
6. Epithelial cell junctions Kelly BéruBé, Toxicology 2010
7. Epithelial cell junctions http://antranik.org/detailed-features-of-epithelia
8. Drosophila development http://www.youtube.com/watch?v=Lb6TJzTLg_E
9. The paper: an overview What is driving tissue invagination in the dorsal folds? early gastrula late gastrula Fig. 1a-b
10. The paper: an overview What is driving tissue invagination in the dorsal folds? early gastrula late gastrula Fig. 1a-d
11. The paper : an overview What is driving tissue invagination in the dorsal folds? early gastrula late gastrula Fig. 1e-f
12. The paper : an overview Apical narrowing often precedes tissue invagination L. Rhumbler (1902)
13. The paper : an overview Apical narrowing often precedes tissue invagination L. Rhumbler (1902)
14. The paper : an overview Folding of the dorsal epithelium is no exception Supplementary Fig. 2
15. The paper : an overview Folding of the dorsal epithelium is no exception Supplementary Fig. 2
16. The paper : an overview Folding of the dorsal epithelium is no exception Supplementary Fig. 2
17. The paper : an overview The canonical mode of apical constriction
18. The paper : an overview The canonical mode of apical constriction
19. The experiment: an overview The canonical mode of apical constriction Nature 457, 495-499 (22 January 2009)
20. The experiment: an overview The canonical mode of apical constriction Nature 457, 495-499 (22 January 2009)
21. The experiment: an overview The canonical mode of apical constriction Nature 457, 495-499 (22 January 2009)
22. The paper : an overview Myosin, however, does not appear to drive dorsal folding Nature 457, 495-499 (22 January 2009)
23. The paper : an overview Myosin, however, does not appear to drive dorsal folding Then what is? Supplementary Fig. 3
24. The paper : an overview Junctional positioning and Bazooka:Par-1 Supplementary Fig. 13
25. The paper : an overview Schematic model for dorsal fold initiation Supplementary Fig. 13
26. The paper : an overview Schematic model for dorsal fold initiation Supplementary Fig. 13
27. The paper : an overview Schematic model for dorsal fold initiation Supplementary Fig. 13
28. The paper : an overview Schematic model for dorsal fold initiation Supplementary Fig. 13
29. The paper : an overview Junctional positioning and Bazooka:Par-1 Cur. Cell Biology 23:540-546 (2011)
30. The paper : an overview Bazooka position correlates with Bazooka:Par-1 Bazooka Par-1 Fig. 2d (modified)
31. The paper : an overview Junctions in the epithelium are mechanically coupled by an actin belt
32. The paper : an overview Par-1 is localized basally Bazooka is localized subapically
33. The paper : an overview As Par-1 levels decrease Bazooka can expand basally
34. The paper : an overview As Par-1 levels decrease Bazooka can expand basally
35. The paper : an overview aPKC establishes the apical domain, enabling junctional disassembly at the apical margin
36. The paper : an overview aPKC establishes the apical domain, enabling junctional disassembly at the apical margin
37. The paper : an overview aPKC establishes the apical domain, enabling junctional disassembly at the apical margin
39. Who does?
40. Neural tube closure is a remarkable example of coordinated tissue movements Curr Biol. 2012 Jul 24;22(14):R574-6.
41. Curr Biol. 2012 Jul 24;22(14):R574-6. Neural tube closure is a remarkable example of coordinated tissue movements
42. Curr Biol. 2012 Jul 24;22(14):R574-6. Neural tube closure is a remarkable example of coordinated tissue movements
43. More than 100,000 babies are born each year with neural tube defects such as anencephaly and spina bifida Curr Biol. 2012 Jul 24;22(14):R574-6.
44. Curr Biol. 2012 Jul 24;22(14):R574-6. More than 100,000 babies are born each year with neural tube defects such as anencephaly and spina bifida The introduction of folic acid supplements has significantly reduced the prevalence of these conditions, yet neural tube defects remain a leading cause of congenital disorders
45. More than 100,000 babies are born each year with neural tube defects such as anencephaly and spina bifida Curr Biol. 2012 Jul 24;22(14):R574-6. The introduction of folic acid supplements has significantly reduced the prevalence of these conditions, yet neural tube defects remain a leading cause of congenital disorders
46. A closer look at the paper
47. The Goal: Identify a dynamic cellular process that precedes cell shape change apical constriction
48. The Goal: Identify a dynamic cellular process that precedes cell shape change apical constriction What they know: Myosin II is not the driver thus, not canonical mode
49. The Goal: Identify a dynamic cellular process that precedes cell shape change What they know: Myosin II is not the driver apical constriction thus, not canonical mode Supplementary Fig. 3
50. The Goal: Identify a dynamic cellular process that precedes cell shape change What they know: Myosin II is not the driver apical constriction thus, not canonical mode Supplementary Fig. 3
51. The Goal: Identify a dynamic cellular process that precedes cell shape change What they know: E-Cad shows a cell-type-specif change in its positioning apical constriction shifts down in ‘initiating’ cells, remains subapical in ‘neighbors’
52. The Goal: Identify a dynamic cellular process that precedes cell shape change What they know: E-Cad shows a cell-type-specif change in its positioning apical constriction shifts down in ‘initiating’ cells, remains subapical in ‘neighbors’ Supplementary Fig. 3
53. The Goal: Identify a dynamic cellular process that precedes cell shape change What they know: E-Cad shows a cell-type-specif change in its positioning apical constriction shifts down in ‘initiating’ cells, remains subapical in ‘neighbors’ Supplementary Fig. 3c
54. The Goal: Identify a dynamic cellular process that precedes cell shape change What they know: E-Cad shows a cell-type-specif change in its positioning apical constriction shifts down in ‘initiating’ cells, remains subapical in ‘neighbors’ gastrulation Fig. 1h
55. The Goal: Identify a dynamic cellular process that precedes cell shape change What they know: E-Cad shows a cell-type-specif change in its positioning apical constriction shifts down in ‘initiating’ cells, remains subapical in ‘neighbors’ Supplementary Fig. 4
56. The Goal: Identify a dynamic cellular process that precedes cell shape change What they know: E-Cad shows a cell-type-specif change in its positioning apical constriction shifts down in ‘initiating’ cells, remains subapical in ‘neighbors’ Supplementary Fig. 4
57. The Goal: Identify a dynamic cellular process that precedes cell shape change What they know: Levels of Bazooka and aPKC are not differentially regulated across dorsal epithelum apical constriction Supplementary Fig. 7
58. The Goal: Identify a dynamic cellular process that precedes cell shape change What they know: Par-1 levels in initiating cells drop to 80% of Par-1 levels in neighboring cells apical constriction as gastrulation commences Fig. 2b-c
59. The Goal: Identify a dynamic cellular process that precedes cell shape change What they know: Par-1 levels in initiating cells drop to 80% of Par-1 levels in neighboring cells apical constriction as gastrulation commences The question: Does reduction in Par-1 levels in initiating cells correlate temporally with the junctional shift?
60. The Goal: Identify a dynamic cellular process that precedes cell shape change What they know: Par-1 levels in initiating cells drop to 80% of Par-1 levels in neighboring cells apical constriction as gastrulation commences The question: Does reduction in Par-1 levels in initiating cells correlate temporally with the junctional shift? The plan: Quantify levels of Par-1 in fixed embryos and determine the position of junctions using Bazooka staining
61. The dynamics of Bazooka and Par-1 during dorsal fold initiation Does Par-1 downregulation allow Bazooka to localize more basally? Fig. 2
62. Uniform reduction of Par-1 by RNAi The junctional positioning is uniform across the entire dorsal epithelium and the initiation of dorsal folds is abolished despite the normal appearance of junction and epithelial structure Fig. 3b
63. Uniform reduction of Par-1 by RNAi Thus, dorsal fold formation seems to require a differential positioning of junctions between the initiating cells and their neighbours Fig. 3b
64. What they know: Par-1 phosphorylates and thereby excludes Bazooka from the basal- lateral regions of polarized epithelial cells The plan: Introduce a mutant form of Bazooka that cannot be phosphorylated by Par-1
65. BazookaS151A, S1085A–GFP Dorsal fold formation is blocked Fig. 3d
66. Altering the ratio of Bazooka:Par1 A uniform increase in Bazooka across the dorsal epithelium Fig. 3e
67. Altering the ratio of Bazooka:Par1 A uniform increase in Bazooka across the dorsal epithelium Supplementary Fig. 10a
68. Altering the ratio of Bazooka:Par1 A uniform increase in Bazooka across the dorsal epithelium Supplementary Fig. 11c
69. What they know: aPKC phosphorylates Bazooka and becomes segregated to establish the apical domain above the junctions The plan: Observe what happens in embryos that lack aPKC activity
70. Altering the level of aPKC Loss of aPKC results in an expansion of the junctional domain and a failure to shorten the initiating cells Initiating cell Neighboring cell Fig. 4a
71. Altering the level of aPKC Loss of aPKC results in an expansion of the junctional domain and a failure to shorten the initiating cells Fig. 4a-b
72. Altering the level of aPKC It seems that the segregation of aPKC from Bazooka establishes the apical domain, enabling junctional disassembly at the apical margin of the junctions. Fig. 4a-b
73. To summarize Junctions in the epithelium are mechanically coupled by an actin belt
74. To summarize Par-1 is localized basally Bazooka is localized subapically
75. To summarize As Par-1 levels decrease Bazooka can expand basally
76. To summarize As Par-1 levels decrease Bazooka can expand basally
77. To summarize aPKC establishes the apical domain, enabling junctional disassembly at the apical margin
78. To summarize aPKC establishes the apical domain, enabling junctional disassembly at the apical margin
79. To summarize aPKC establishes the apical domain, enabling junctional disassembly at the apical margin
80. Bazooka localization During cellularization, three mechanisms normally position Baz: (1) an apicolateral actin-based scaffold, (2) basal-toapical dynein-based transport, and (3) an undefined mechanism evident from analysis of dynein mutants in which basally mislocalized Baz redistributes apically by gastrulation (Harris and Peifer, 2005).
81. Bazooka localization