Early Vert Development (3) Flashcards
Why was noggin believed to be the neural inducing factor?
- it was expressed at the right place at the right time
- expressed in dorsal lateral area, prechordal mesoderm and dorsal mesoderm
What gain-of-function experiment was done to see if noggin can function as a neural inducer?
- 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
What is neural induction also called? What is it mediated by?
- dorsalization of the ectoderm
- mediated by secreted BMP inhibitors from dorsal mesoderm
What loss-of-function experiment was conducted to see if noggin can function as a neural inducer?
- 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
What is Sox2?
- a marker of neural development
What other factors were identified that can antagonize BMP and are localized in the same areas?
- follistatin
- chordin
- xnr3
- cerberus
- creates functional redundancy
What experiment could be conducted to see if these other factors are necessary for neural development?
- 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
Why is there redundancy?
- 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
What characteristics does the blastula have?
- 3 germ layers are specified
- layers can act on each other to induce development
- blastocoel
What other experiments can be done to investigate whether inductive interactions occur between other germ layers?
- remove mesoderm and see if ectoderm and endoderm have an effect
- result: ectoderm becomes mesoderm
- indicates that endoderm has mesoderm inducing activity
What is “kitchen sink” science?
- sometimes no good rationale or hypothesis but something interesting is found
What is known about exposing a 1 cell embryo to LiCl?
- became “dorsalized”
- ectopic neural
- lots of notochord
- dorsal mesoderm
What is known about exposing a 1 cell embryo to UV irradiation?
- became “ventralized”
- no neural tissue
- no organizer
- lots of ventral mesoderm (eg blood)
What does the LiCl and UV irradiation kitchen sink science indicate?
- there is a dorsal and ventral difference
- may be some factor present in the dorsal endoderm that is important for dorsal mesoderm
What observations have we gathered so far?
- endoderm can induce mesoderm
- UV kills organizer formation and dorsal development
- still get ventral endoderm and ventral mesoderm
What question and experiment might we do for the hypothesis “the dorsal endoderm has a factor that can induce the organizer”?
- 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
What results were found from the rescue experiment on UV ventralized embryos?
- transplanted the dorsal vegetal blastomere from a normal embryo to the UV treated embryo
- rescue was successful
What conclusions can be made about the inductive capacity of the endoderm?
- endoderm can induce mesoderm
- some endoderm (Nieuwkoop centre) is specialized to induce dorsal mesoderm (Spemann organizer)
What is the Nieuwkoop centre?
- endodermal region that can induce dorsal mesoderm (Spemann organizer)
What vegetally-localized proteins/mRNAs were identified that might be important for specifying endoderm fate? How can we examine these closer?
- VegT: T-box transcription factor
- GSK-3 binding protein
- loss of function or gain of function
What general experiment was done to examine VegT?
- use a VegT antisense oligos injected into unfertilized egg
- result: a blob of tissue
- only tells you that you screwed up development!
What is needed in the VegT experiment to make a better assay?
- markers for endoderm and mesoderm to examine effect on these tissues
What markers were used in the VegT experiment?
- Mxr: pan-endodermal marker
- Xbra: pan-mesodermal marker
- then used RT-PCR to look at the markers
What are the results of the VegT (marker) experiment?
- 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)
What do the VegT results indicate?
- VegT may have an important role in endoderm and consequently in mesoderm formation
What is the effect on the wnt pathway when GSK-3 binding protein (GBP) is added?
- GBP inhibits GSK-3 which releases the inhibition on b-catenin
- wnt pathway is turned on
Where is beta catenin localized?
- beta-catenin nuclear localization in dorsal region of blastula
Where is GBP likely localized? What hypothesis does this lead to?
- 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
What happens when GBP mRNA is injected into a UV damaged embryo?
- get a rescue of dorsal development
Where is GBP localized in the unfertilized egg?
- 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
How might the GBP be translocated?
- perhaps the sperm plays a role since it causes asymmetry
- cytoplasmic cortex rotational shift
In general, what are microtubules?
- polarized structures
- + end is where mt’s elongate/grow
How do microtubules begin forming in the egg?
- when the sperm enters, a centriole (microtubule organizing center) is introduced
- microtubules start growing outwards from the centrioles
What is seen with tubulin immunolabeling?
- 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
Based on the microtubule correlative data, what might we hypothesize about GBP?
- GBP might be shuttled along the microtubules
- since microtubule binding motor proteins shuttle proteins and vesicles along microtubules
What experiment can be done to examine the relationship between microtubules and GBP?
- 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
What can be stated about GBP movement?
- 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
