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BIOL 309 > Early Vert Development (3) > Flashcards

Early Vert Development (3) Flashcards

1
Q

Why was noggin believed to be the neural inducing factor?

A
  • it was expressed at the right place at the right time

- expressed in dorsal lateral area, prechordal mesoderm and dorsal mesoderm

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2
Q

What gain-of-function experiment was done to see if noggin can function as a neural inducer?

A
  • take a part of animal cap and grow in isolation: becomes epidermis
  • take a part of animal cap and grow with noggin: becomes neural tissue
  • this indicates noggin is sufficient
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3
Q

What is neural induction also called? What is it mediated by?

A
  • dorsalization of the ectoderm

- mediated by secreted BMP inhibitors from dorsal mesoderm

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4
Q

What loss-of-function experiment was conducted to see if noggin can function as a neural inducer?

A
  • knockout experiment
  • injection of antisense RNA to knock-down Noggin at Xenopus 2-cell stage
  • assay: in situ hybridization for Sox2 at the neural plate stage
  • result: no effect, because there may be other factors that are doing the same thing (redundancy)
  • indicates noggin is not necessary
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5
Q

What is Sox2?

A
  • a marker of neural development
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6
Q

What other factors were identified that can antagonize BMP and are localized in the same areas?

A
  • follistatin
  • chordin
  • xnr3
  • cerberus
  • creates functional redundancy
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7
Q

What experiment could be conducted to see if these other factors are necessary for neural development?

A
  • knock down more factors and see if neural tissue is developed
  • with three knockdowns (noggin, folistatin, chordin), neural tissue is reduced
  • individual knockdowns give no effect
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8
Q

Why is there redundancy?

A
  • as a backup
  • to give a more robust signal
  • genes are often highly related to each other (could be from gene duplication)
  • not exactly known for sure
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9
Q

What characteristics does the blastula have?

A
  • 3 germ layers are specified
  • layers can act on each other to induce development
  • blastocoel
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10
Q

What other experiments can be done to investigate whether inductive interactions occur between other germ layers?

A
  • remove mesoderm and see if ectoderm and endoderm have an effect
  • result: ectoderm becomes mesoderm
  • indicates that endoderm has mesoderm inducing activity
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11
Q

What is “kitchen sink” science?

A
  • sometimes no good rationale or hypothesis but something interesting is found
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12
Q

What is known about exposing a 1 cell embryo to LiCl?

A
  • became “dorsalized”
  • ectopic neural
  • lots of notochord
  • dorsal mesoderm
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13
Q

What is known about exposing a 1 cell embryo to UV irradiation?

A
  • became “ventralized”
  • no neural tissue
  • no organizer
  • lots of ventral mesoderm (eg blood)
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14
Q

What does the LiCl and UV irradiation kitchen sink science indicate?

A
  • there is a dorsal and ventral difference

- may be some factor present in the dorsal endoderm that is important for dorsal mesoderm

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15
Q

What observations have we gathered so far?

A
  • endoderm can induce mesoderm
  • UV kills organizer formation and dorsal development
  • still get ventral endoderm and ventral mesoderm
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16
Q

What question and experiment might we do for the hypothesis “the dorsal endoderm has a factor that can induce the organizer”?

A
  • Does UV treatment kill a dorsal endoderm factor required to induce the organizer
  • can you rescue UV ventralized embryos with a normal dorsal endoderm cell
17
Q

What results were found from the rescue experiment on UV ventralized embryos?

A
  • transplanted the dorsal vegetal blastomere from a normal embryo to the UV treated embryo
  • rescue was successful
18
Q

What conclusions can be made about the inductive capacity of the endoderm?

A
  • endoderm can induce mesoderm

- some endoderm (Nieuwkoop centre) is specialized to induce dorsal mesoderm (Spemann organizer)

19
Q

What is the Nieuwkoop centre?

A
  • endodermal region that can induce dorsal mesoderm (Spemann organizer)
20
Q

What vegetally-localized proteins/mRNAs were identified that might be important for specifying endoderm fate? How can we examine these closer?

A
  • VegT: T-box transcription factor
  • GSK-3 binding protein
  • loss of function or gain of function
21
Q

What general experiment was done to examine VegT?

A
  • use a VegT antisense oligos injected into unfertilized egg
  • result: a blob of tissue
  • only tells you that you screwed up development!
22
Q

What is needed in the VegT experiment to make a better assay?

A
  • markers for endoderm and mesoderm to examine effect on these tissues
23
Q

What markers were used in the VegT experiment?

A
  • Mxr: pan-endodermal marker
  • Xbra: pan-mesodermal marker
  • then used RT-PCR to look at the markers
24
Q

What are the results of the VegT (marker) experiment?

A
  • control: increase of Mxr and Xbra following gastrulation (meaning mesoderm and endoderm forming)
  • VegT kd: no expression of Mxr or Xbra (meaning endoderm and mesoderm not developing)
25
Q

What do the VegT results indicate?

A
  • VegT may have an important role in endoderm and consequently in mesoderm formation
26
Q

What is the effect on the wnt pathway when GSK-3 binding protein (GBP) is added?

A
  • GBP inhibits GSK-3 which releases the inhibition on b-catenin
  • wnt pathway is turned on
27
Q

Where is beta catenin localized?

A
  • beta-catenin nuclear localization in dorsal region of blastula
28
Q

Where is GBP likely localized? What hypothesis does this lead to?

A
  • since beta catenin is localized to the dorsal side, GBP is likely to be as well
  • hypothesis: GBP is a factor necessary for inducing dorsal endoderm/mesoderm
29
Q

What happens when GBP mRNA is injected into a UV damaged embryo?

A
  • get a rescue of dorsal development
30
Q

Where is GBP localized in the unfertilized egg?

A
  • GBP is localized in the vegetal pole uniformly
  • despite thinking it may be localized in future dorsal region so it must move there at some point
  • GBP and disheveled are translocated to the dorsal side of the egg during ferilization
31
Q

How might the GBP be translocated?

A
  • perhaps the sperm plays a role since it causes asymmetry

- cytoplasmic cortex rotational shift

32
Q

In general, what are microtubules?

A
  • polarized structures

- + end is where mt’s elongate/grow

33
Q

How do microtubules begin forming in the egg?

A
  • when the sperm enters, a centriole (microtubule organizing center) is introduced
  • microtubules start growing outwards from the centrioles
34
Q

What is seen with tubulin immunolabeling?

A
  • microtubules are seen in red and shortly after fertilization they begin to organize themselves in long parallel bundles with the same polarity
  • microtubule array network
  • mirotubules grow towards the future dorsal side
35
Q

Based on the microtubule correlative data, what might we hypothesize about GBP?

A
  • GBP might be shuttled along the microtubules

- since microtubule binding motor proteins shuttle proteins and vesicles along microtubules

36
Q

What experiment can be done to examine the relationship between microtubules and GBP?

A
  • correlative data: direct approach, look and see
  • engineered a GBP-GFP fusion protein expression construct
  • injected GBP-GFP encoding RNA into unfertilized eggs
  • fertilize the egg and observe the GBP movement
  • can see that they are clustering and moving in the same direction
37
Q

What can be stated about GBP movement?

A
  • GBP (and disheveled and wnt11 mRNA) undergo cortical/cytoplasmic translocation in the fertilizing egg
  • they are carried by kinesin along the microtubules to the + end

BIOL 309 (14 decks)

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