- TAPE 1
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Oocyte and Early Embryonic Manipulations
1. Oocyte isolation from dissected ovary; germinal vesicle isolation (isolation of ovary from adult frogs is discussed in Tape #3) (R. Elinson)
2. Oocyte isolation from dissected ovary (J. Heasman)
3. Isolation of cortex from fertilized eggs (R. Elinson)
4. Analysis of axial defects in embryos treated with U.V. light or LiCl (R. Elinson)
5. Observation of pigment differences in 4-cell embryos indicative of the presumptive dorsal/ventral axis; injection of 4-cell embryos (J. Gurdon)
6. “Tipping and staining” method for determining the dorsal/ventral axis of 1-cell embryos (T. Doniach)
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Basic Manipulations of Blastula, Gastrula, and Early Neurula Embryos
7. Removal of membranes from embryos; isolation of animal caps from blastula embryos; examination of mesodermal induction by recombinants of animal and vegetal tissue (J. Gurdon)
8. Dissociation of animal caps and reaggregation of cells; implanting cells into animal cap sandwiches (J. Gurdon)
9. Animal cap isolation; dorsal/ventral pigment and cell-size differences in blastula animal region; transplanting ectodermal piece from one cap to another (J. Smith)
10. Dissection of embryos into animal, marginal, and vegetal regions; mesoderm induction in animal/vegetal recombinants (J. Smith)
11. Rapid isolation of animal caps (J. Smith)
12. Isolation of animal caps by rapid movements of eyebrow hair knives (R. Harland)
13. Isolation of animal caps from albino embryos; further illustration of use of eyebrow hair to cut animal caps (J. Smith)
14. Spemann Organizer graft (J. Slack)
15. Animal cap isolations; mesoderm induction in animal/vegetal recombinants (J. Slack)
16. Use of trypsin to isolate tissue rudiments from neurula-stage embryos (J. Slack)
17. Dissection of epidermis from neurula-stage embryos (J. Slack)
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Gastrula Stages: Detailed Observations and Dissections
18. Introduction; use of microscope; strategies for dissection (R. Keller)
19. Isolation of small animal cap pieces and epithelial layer; dissection technique and strategies for dissection (R. Keller)
20. Tour of the gastrula-stage embryo, external features (R. Keller)
- TAPE 2
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Gastrula Stages: Detailed Observations and Dissections (continued from Tape 1)
21. Tour of the gastrula stage embryo, internal features; skewering technique for identifying anterior/posterior position on the dorsal axis; manipulating dorsal tissues (R. Keller)
22. Examining stage 10.5-11 embryos; identifying dorsal vs. ventral sides of the embryo (R. Keller)
23. Examining stage11-11.5 embryos; detailed look at stage 10.5 embryos (R. Keller)
24. Caution on identifying dorsal and ventral sides of stage 11 embryos (R. Keller)
25. Description of stage 10 embryos; cautions on using pigmentation for identifying dorsal/ventral differences; Keller sandwich (R. Keller)
26. Keller sandwich II (R. Keller)
27. Keller sandwich III (R. Keller)
28. Observation of Keller sandwiches after culture to early neurula stages (R. Keller)
29. Observation of Keller sandwiches after culture to early neurula stages (approximately 1 hr later than previous segment) (R. Keller)
30. Observation of Keller sandwiches after culture to later neurula stages (R. Keller)
31. Critical evaluation of successful and unsuccessful Keller sandwiches and “giant” sandwiches (R. Keller)
32. Preparation of Keller sandwich (T. Doniach)
33. Preparation of “giant” sandwich (includes entire marginal zone) (R. Keller)
34. Preparation of “pita sandwich (includes entire animal and marginal zone folded over on itself); observation of dorsal pigmentation in animal cap cells (R. Keller)
35. Preparation of Keller sandwich preserving head mesoderm in the recombinant (R. Keller)
36. Preparation of “lap” joint for examining signaling by planar induction (R. Keller)
37. “Harris flip” technique for cutting tissue; boundary of involuting and noninvoluting tissues; determining extent of convergence and extension along the anterior/posterior axis (R. Keller)
38. Observing the leading edge of involuting dorsal endoderm/mesoderm (R. Keller)
39. Description and dissection of dorsal tissues from stage 11.5-12 embryos; begin dissection of dorsal tissues from stage 13 embryos (R. Keller)
- TAPE 3
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Gastrula Stages: Detailed Observations and Dissections (continued from Tape 2)
40. Finish dissection of dorsal tissues from stage 13 embryos (R. Keller)
41. Discussion of tendencies of Keller explants to form secondary vertical interactions (R. Keller)
42. Observation of notochord, head mesoderm in cultured Keller explants (R. Keller)
43. Result of anteroposterior reversal of marginal zone; virtually any manipulation is possible with Xenopus embryos (R. Keller)
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Specialized Operations on Gastrula and Neurula Stage Embryos
44. Implanting dorsal mesoderm into the blastocoel of a host embryo (“Einsteck” experiment) (T. Doniach)
45. Recombinant of dorsal mesoderm and animal cap ectoderm illustrating identification of different anteroposterior regions of dorsal mesoderm and their isolation for recombinant experiments (T. Doniach)
46. Recombinant of dorsal mesoderm and animal cap ectoderm; observation after culture to neural tube stages (R. Keller)
47. “Einsteck” procedure; placing dorsal mesoderm into the blastocoel of a host embryo; illustration of clay dishes for dissections; identification of dorsal side of stage 11 embryos (R. Grainger)
48. Recombinant of neural plate (isolated by trypsinization) and animal cap; another method for animal cap isolation; using clay dishes for dissections (R. Grainger)
49. Transplanting animal cap ectoderm to the lateral region of a neural-plate stage host embryo; illustration of use of glass bridges to hold tissues in place (R. Grainger)
50. Isolation of presumptive lens ectoderm from stage 14 embryo; discussions of separation of tissue layers and the use of clay dishes for dissection (R. Grainger)
51. Isolation of forebrain of stage 23 embryo following trypsin treatment (R. Grainger)
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Genetic Manipulation of Xenopus Embryos, Including Transgenesis
52. Dissection of ovary from adult Xenopus, as required for the genetic manipulations discussed in the next demonstration (suturing of female after surgery is also illustrated in the next section) (R. Elinson)
53. Implanting oocytes into adult Xenopus; a method for assaying embryos developing from oocytes treated to alter gene expression (e.g. with anti-sense reagents); suturing female frog after implanting oocytes (or after removal of ovary) (J. Heasman)
54. Demonstration of the classical nuclear transplantation technique (J. Gurdon)
55. Needles used for nuclear transplantation into embryos compared to needles used for RNA injections (E. Amaya)
56. Observation of flow from nuclear transplant needle for injecting sperm nuclei into eggs; technique for injecting nuclei into eggs; how to screen embryos to ascertain the number of nuclei injected; observing cortical contraction after egg activation (E. Amaya)
57. Jab technique for nuclear injections (E. Amaya)
58. Observation at the time of first cleavage of embryos injected with sperm nuclei (E. Amaya)
59. Preparation of testis for sperm isolation prior to nuclear transplantation (K. Kroll)
60. Maceration of testis for sperm isolation prior to nuclear transplantation (K. Kroll)
61. Normal and swelled sperm (used in DNA integration experiments prior to nuclear transplantation) viewed by fluorescence microscopy (K. Kroll)
62. Preparing microinjection needle points for nuclear transplantation (K. Kroll)
63. Injecting sperm nuclei into embryos: illustration of injection technique; observation of activated embryos (K. Kroll)
64. Further illustration of nuclear injection technique (K. Kroll)
65. Distinguishing normal and abnormal embryos the day after nuclear transplantation (K. Kroll)
66. Further discussion of healthy and unhealthy embryos following nuclear transplantation (K. Kroll)
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Pushing the Envelope
67. Skinning a gastrula embryo: try that with zebrafish embryos (R. Keller)
68. Removing the vitelline envelope from embryos with 12-inch forceps: the limits of dissection (R. Grainger)
