Xenopus development

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  1. What kind of yolk does it have? What kind of cleavage?
    • Mesolesical yolk, yolk is on vegetal side mainly, but holoblastic cleavage
    • the yolk part takes longer to split though so the cells there are bigger because they divide less
  2. point where sperm makes contact is?
  3. what are the beginning events of cleavage/gastrulation in xenopus?
    sperm enters animal pole- sets up D/V axis - sperm centriole organizes microtubules to be parallel in the vegetal cytoplasm separating the cortical cytoplasm - cortical cytoplasm rotates 30 degrees and creates grey crescent
  4. where is and what is important about the grey crescent?
    it is located directly across the sperm entry point and is the place where gastrulation begins!
  5. what is different about formation of the archenteron in the frog and the sea urchin?
    • sea urchin - it happens in the vegetal pole
    • frog - it happens in the marginal zone (right between the animal and the vegetal)
  6. what are the sizes of cells in the vegetal and animal zones resulting from cleavage?
    • cells in vegetal - bigger and fewer
    • cells in animal - smaller and more (bc more divisions)
  7. how many cells constitutes a morula?
    what about a blastula?

    128 cells (when blastocoel becomes apparent)
  8. what are the 3 axis that are set up at fertilization?
    • anterior - posterior
    • dorsal - ventral
    • lateral
  9. what is the funciton of the blastocoel?
    • permits cell migration during gastrulation
    • prevents cell- cell interactions that arent supposed to happen yet
  10. depp layers give rise to?
    superficial layers give rise to?
    • deep - mesoderm cells
    • superficial - endoderm and ectoderm
  11. where is the marginal zone?
    between vegetal half and animal half
  12. what are the 3 main movements that happen at the beginning of gastrulation?
    1) dorsal side/grey crescent - invaginate forming the dorsal blastopore lip (cells are called bottle cells) - forms the archenteron (mesoderm)

    2) marginal zone cells involute

    3) animal cells - epiboly
  13. what happens to the cells that compose the dorsal lip?
    • as they enter the embryo, they change their fate, so the first cells - head mesoderm (prechordal plate)
    • 2nd cells - chordamesoderm, foot of archenteron, notochord
  14. what happens to the dorsal mesoderm?
    what happens to the dorsal lip?
    what happens to the blastocoel?
    • extends anteriorly due to convergent extension
    • expands laterally and ventrally
    • gets displaced to the ventral side
  15. what happens to the dorsal lip?
    expands laterally and ventrally
  16. what is the yolk plug?
    remaining patch of endoderm, all the other endoderm is in the middle already
  17. whats the order of the lip formations?
    dorsal, lateral, and ventral
  18. describe what happens when the ectoderm does epiboly
    • the yolk plug (endo) stays in place and the ectoderm epibolys while the mesoderminvolutes
    • increase in cell #
    • integration of several layers into one
  19. what happens if you prevent 30 degree rotation?
    • no dorsal axis!
    • gastrulation occurs and forms 3 germ layers, but only most ventral mesoderm forms
  20. what did Hans Spemman do?
    took a baby hair from his daughter as a "lasso" and cleaved an early embryo... found out that if cell gets part of grey crescent then you get a normal embryo, but if not grey crescent abnormal embryo (lacks dorsal structures)--> gray crescent is key!

    also... 1) EARLY GASTRULA- switched neuro ectoderm and epidermis cells/locations and they naturally switched, but LATE GASTRULA - he switched them and got 2 neural plats (movements during gastrulation were important in cell fate decisions)
  21. project of Spemann and Hilde Mangold?
    inserted dorsal lip of the blastopore tissue into what was supposed to become belly skin and it acted autonomously! = ORGANIZER b/c it organized the surrounding tisues
  22. what are the functions of the organizer?
    differentiate autonomously AND organize tissues around it (all 3 axis) - directly across where sperm enters - this is the point where two signals converge!
  23. what is are the 2 signals that converge?
    • 1- be dorsal
    • 2 - be mesoderm
  24. nieuwkoop experiment?
    mix animal and vegetal cap cells together - animal cells converted to mesoderm by factors released from vegetal cells
  25. 64 cell embryo experiment relating to organizing center:
    1) inject most dorsal vegetal cells into ventral most vegetal region of another embryo gives you a new gastrulation site

    2) vegetal blastomeres form a 32 cell embryo, placed next to animal cap - dorsal most vegetal cells = Nieuwkoop center
  26. what does the Newkoop center do?
    tells the organizing center to be an organizer
  27. what is the WNT pathway?
    • wnt binds to receptor called frizzled protein - which frees disheveled - blocks GSK3 (which normally targets B catenin for destruction)
    • B catenin - turns on transcription factors (LEF/TCF) that bind to promoters and turn on gene expression
  28. how is Beta catenin oriented in the embryo?
    what happens if you inactivate GSK3?
    • starts out everywhere, but it is turned off on the ventral side, so only expressed on the dorsal side (like a gradient)
    • - then you get a double axis b/c body thinks it has two dorsal sides
  29. how does disheveled stabilize b catenin?
    dsh starts out in cortex of vegetal plate, then rotation happens 30 degrees, Dsh + wnt mrna + GBD --> inactivate GSK3 B
  30. what are all 4 proteins that turn on transcription of organizer genes?
    simwa + twin + TGF - beta + Smad2/4
  31. what is the generic mesoderm signal? and what turns it on?
    Xbra gets turned on by Vg1 and VegT
  32. what is vg1 and what is vegT?
    vg1 = TGF beta family member (secreted by vegetal cells)

    vegT = transcription factor - makes TGF beta family members
  33. how do the gradients determine what type of mesoderm will become what?
    • B catenin coming in from the dorsal side
    • VegT and Vg1 coming in from the vegetal side
    • where they overlap is the Newcoop center
    • - get a gradient of Xnr (nodal related) that increases as you get more dorsal, diff amounts of Xnr makes diff things

    ALSO get a gradient of BMP4 and Xwnt8 that decreases as you go more dorsal
  34. what amounts of Xnr give you what?
    • high - organizer
    • intermediate = lateral mesoderm
    • low = ventral mesoderm
  35. what are the properties of the primary organizer tissue?
    • ability to become dorsal mesoderm
    • ability to dorsalize surrounding mesoderm into lateral mesoderm
    • ability to dorsalize ectoderm into neural ectoderm
    • ability to initiate movement of gastrulation
    • ability to cause neural plate to become neural tube
  36. cells of the organizer can give rise to:
    • pharyngeal endoderm (induces forebrain and midbrain)
    • head mesoderm (same as above)
    • dorsal mesoderm (primarily notochord) - induces hindbrain and trunk
    • dorsal blastopore lip (induces tip of tail)
  37. experiments that indicate orgnanizer proteins are dosage dependent:
    if you hit an egg with lots of UV light then you dont get cortical rotation, but you can rescue it if you inject NOGGIN, but if you inject too much noggin then you only get dorsal
  38. what does BMP do?
    says "be epidermis and ventral!"
  39. how do the secreted organizer molecules organize?
    they are BMP blockers! so they dont say be neural but they say DONT BE VENTRAL!
  40. what happens if you dont block BMP?
    then everything will become ventral
  41. what are some of the organizer proteins? and their fx?
    chordin, noggin, follistatin, Xnr3 - they bind and inactivate BMP4
  42. in what fashion does BMP4 turn on gene transcription?
    sometimes only a little is needed, sometimes mod, sometimes a lot is needed to turn on genes
  43. what did Etienne Geoffroy Saint - Hilaire say about the lobster? what does this say about D/V process?
    • that instead of having a dorsal cord it had a ventral cord, SO EVERYTHING IS ACTUALLY FLIPPED!
    • same molecules but on the opposite sides. for example: cordin is a BMP blocker, so in this case it would be located in the ventral most tissue and there would be high BMP on the dorsal side
    • - it is highly conserved!
  44. explain what regional specificity of induction is/ otto mangold's experiment?
    • he took different parts of the archenteron (early dorsal lip/organizer cells = anterior archenteron) and transplanted them into early embryo and got double axis only on anterior part of frog though
    • - transplant late dorsal cells (end of archenteron) and got a double axis down by the trunk (posterior part)
  45. what happens when you transplant the WHOLE DORSAL LIP (early on) into an embryo?
    get a FULL double axis
  46. what is regional specificity?
    anterior - posterior
  47. how does the anterior -posterior axis get set up? describe the gradient...
    • the gradient of BMP4 also has a gradient of Wnt
    • there are Wnt inhibitors that block Wnt and play a role in the EARLY dorsal cells = makes ANTERIOR!
  48. what are the Wnt inhibitors and what secretes them?
    • cerberus (3 headed dog)
    • Frzb
    • Dickkopf

    - organizer secretes them
  49. block BMP and Wnt?
    block just BMP?
    block neither?
    • - dorsal/anterior = HEAD
    • - dorsal/posterior = trunk
    • - ventral
  50. what happens if you over express ceberus?
    blocks Wnt and BMP, so you get a bunch of heads
  51. how does Frzb function?
    • it is the soluble form of frizzled, competes with frizzled by binding to Wnt
    • expressed only in anterior organizer tissue
  52. what happens if you inject Frzb mRNA into marginal zone cells?
    inhibits trunk formation ah!
  53. what is the linear series of the dorsal organizing tissue?
    • pharyngeal endoderm is first (most anterior endoderm), then prechordal plate mesoderm (most anterior mesoderm), then notochordal mesoderm
    • - study slide on 2nd to last page of chap 7 handout
  54. break it down big pic/time:
    • 1) set up niewkoop center (overlap of dorsal and vegetal signals) - from Vg1 and VegT (say vegetal) and B catenin (says be dorsal)
    • 2) niewkoops job is then to set up the organizer tissue (simois and twin expressed in NK + TGF beta and smad - turns - activatess goosecoid (turns on BMP blockers) gradient is set up, which says be organizer tissue)
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Xenopus development
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