Early Vert Development (3)

Question 1

Why was noggin believed to be the neural inducing factor?

Answer 1

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

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

Question 2

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

Answer 2

• 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

Question 3

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

Answer 3

• dorsalization of the ectoderm

- mediated by secreted BMP inhibitors from dorsal mesoderm

Question 4

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

Answer 4

• 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

Question 5

What is Sox2?

Answer 5

• a marker of neural development

Question 6

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

Answer 6

• follistatin
• chordin
• xnr3
• cerberus
• creates functional redundancy

Question 7

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

Answer 7

• 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

Question 8

Why is there redundancy?

Answer 8

• 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

Question 9

What characteristics does the blastula have?

Answer 9

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

Question 10

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

Answer 10

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

Question 11

What is “kitchen sink” science?

Answer 11

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

Question 12

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

Answer 12

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

Question 13

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

Answer 13

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

Question 14

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

Answer 14

• there is a dorsal and ventral difference

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

Question 15

What observations have we gathered so far?

Answer 15

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

Question 16

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

Answer 16

• 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

Question 17

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

Answer 17

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

Question 18

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

Answer 18

• endoderm can induce mesoderm

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

Question 19

What is the Nieuwkoop centre?

Answer 19

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

Question 20

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

Answer 20

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

Question 21

What general experiment was done to examine VegT?

Answer 21

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

Question 22

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

Answer 22

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

Question 23

What markers were used in the VegT experiment?

Answer 23

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

Question 24

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

Answer 24

• 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)

Question 25

What do the VegT results indicate?

Answer 25

- VegT may have an important role in endoderm and consequently in mesoderm formation

Question 26

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

Answer 26

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

Question 27

Where is beta catenin localized?

Answer 27

- beta-catenin nuclear localization in dorsal region of blastula

Question 28

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

Answer 28

- 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

Question 29

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

Answer 29

- get a rescue of dorsal development

Question 30

Where is GBP localized in the unfertilized egg?

Answer 30

- 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

Question 31

How might the GBP be translocated?

Answer 31

- perhaps the sperm plays a role since it causes asymmetry | - cytoplasmic cortex rotational shift

Question 32

In general, what are microtubules?

Answer 32

- polarized structures | - + end is where mt's elongate/grow

Question 33

How do microtubules begin forming in the egg?

Answer 33

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

Question 34

What is seen with tubulin immunolabeling?

Answer 34

- 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

Question 35

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

Answer 35

- GBP might be shuttled along the microtubules | - since microtubule binding motor proteins shuttle proteins and vesicles along microtubules

Question 36

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

Answer 36

- 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

Question 37

What can be stated about GBP movement?

Answer 37

- 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