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2011_Questions_Week_6_Answers - Cell 388 1 The following...

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Unformatted text preview: Cell 388 1. The following picture is taken from a paper that uses an antibody to detect a sub-population of neural crest cells (the antibody is detected with a secondary green fluorescent antibody). The image shows a cross-section through the trunk of an embryo. A) Label the scleretome, neural tube, notochord and epidermis B) What general type of cells will these green cells become? B) Why do you think so? 2. In the following image, increasing amounts of a certain protein are added to neural embryoid bodies (equivalent to neural plate) that are all stained for the presence of Nkx2.2. The top has no protein, the middle has a low amount of this protein, while the bottom image has a higher amount. What protein do you think this is? Why do you need a lot of it? Sonic hedgehog (Shh). You need a lot because Nkx2.2 is one of the most ventral targets of Shh (V3 interneurons). Remember that Shh activates targets in different classes of the ventral neurons in a dose-dependent fashion. V3 interneurons lie right above floorplate and require the second highest doses of Shh protein. mouse genes). hMGFP-positive cells did not stain with an antibody (A-F ) Chi anti-MITF labeled w red). The red blood (B-F) are a The boxed higher ma respective early as st MITF-expr and accum area (MSA melanobla pathway a 20, the ac in the MS observed pathway ( reach the stage 22 ( the ectod cells are fo limb bud of MITF-p ventral m stages. Sc comparable results (see Fig. S2 in against MITF, whereas the rest of the cells in the culture were MITF Although hMGFP was detectable i positive (see Fig. S1 in the supplementary material). control-transfected cultures, hMGFP After confirming the efficacy of the MITF shRNA construct, we observed in the same cell in the shRN used it to knock down expression of MITF in cultured is 3. What is an inhibitory signal in the somite that helps guide trunk neural crest migration? Wherequail it Because MITF functions in melanocy melanoblasts derived from melanoblast clusters. After 24 hours in we assayed some cultures for apoptos expressed? culture, melanoblasts were transfected with either the MITF shRNA S2 in the supplementary material). N construct or with the scrambled control construct. After an additional proliferation were detected. Based on 48 hours, the cells were fixed and stained with the glial marker 7B3 of MITF in cultured melanoblasts s -EphinB1, which is expressed in the posterior somite (Henion et al., 2000) and with anti-MITF antibodies. We also melanocyte fate to a glial phenotype Figure 2. Hedgehog-Dependent Ventralization of Neural Progenitor Cells in Embryoid Bodies immunolabeled with the Hu antibody (a neural marker), but saw no (A) Shh-activated transcriptional pathway of spinal MN generation. Proteins that promote and inhibit MN generation are shown in red and difference in the number of Hu-positive cells. No hMGFP-positive FOXD3 represses expression blue, respectively. (i.e. of HD and bHLH proteins in the caudal neural tube of with the anti-MITF To determine giving rise FOXD3 can (B) Expression MITF shRNA-positive) cells were stainedE9.5 mouse embryos. Progenitors in the domain whether to MNs express Olig2, 4. You want to study neural tube closure in mouse embryos. Nkx6.1,inexpression in of Pax6. antibody and low levels the shRNA-transfected cultures (Fig. 2C,E), compared transfected pFOXD3, which simulta (C) Transcription factor ES cell-derived EBs grown for 3 days in the presence of RA (2 M) alone, and with 10 nM or 1 M with this the hMGFP-negative cells in in real time 65- EGFP, into Hh-Ag1.3. Design an experimental setup that would allow you to monitor 82% of process occurringthe same culture andduring number ofB16-F10 mouse melanom (D) Quantitation oftranscription factor expression in EBs in the presence of RA and Hh-Ag1.3. Mean SEM, lost in cells per section from 69% of the cells (whether hMGFP negative or positive) in the control- was all EGFP-positive cel eight EBs assayed. development. transfected cultures (Fig. 2A,E), indicating that the construct staining with an anti-MITF antibo -You could use the strategy outlined in class lecture, whichsuccessfullyprofile of Hox-c proteintransgeniccontrol-transfectedexamined whether ventral interneuwas to eliminated expression of MITF. In the2001). We therefore has mouse that heterogeneity in staining intensity a tity revealed by the generate a expression Transfec cultures, 26-29% of the cells were 7B3 positive rons are also generated cells were MITF positive.to (Fig. 2B,E). In the (Belting et al., 1998; Liu et al., 2001). in myristoylated YFP (a yellow fluorescent protein that is specifically aexpressed in of the hMGFP-positive cells were You Chx10 V2 neuronsitf (Fig. 3Athe cell membrane). Lhx3RA-treated EBs exposed , SinceshRNA-transfected cultures,ventral interneuShh acts in graded manner, 75% 1 M Hh-Ag1.3. Both repress expression of M and induced at concentrations only slightly below neurons were detected in Lim1/2 cells V0 could then culture mouse embryos and take movies of rons are7B3 positive, compared with 16%under a fluorescent and V1)To determine whether FOXD3 co the processin real time of the hMGFP-negative(likely (Fig. that sufficient for MN generation (Briscoe and Ericson,cells were assayed at 72 and 3H, data not shown). Thus, as these EBs (Figures 3G in avian melanoblasts, cultured quai 2D,E). A similar experiment, in which the microscope. hours and the empty shRNA vector was used as a control, yielded pFOXD3 or with empty pMES and 5. What happens when you electroporate chick embryos with Sox9 in the neural tube? -You get the expression of neural crest markers on the electroporated side of the neural tube - but these cells are unable to migrate out of the neurepithelium 6. Where do neural crest cells form in relation to the neural plate and the epidermis? -They form at the boundary of the neural plate and epidermis. 7. What are the human birth defects corresponding to neural tube closure defects in A) the anterior neural tube and B) the posterior neural tube? -(A) – Anencephal; (B) - Spinal bifida 8. Describe the three primary vesicles of the brain. What will each of them become? -Prosencephalon (Forebrain), Mesencephalon (Midbrain), Rhombencephalon (Hindbrain). 9. In classic experiments, embryologists took naive neural plate and cultured it with notochord tissue to form ventral neuronal fates (including floorplate and motor neurons). What do you predict would happen if you performed these same experiments, substituting notochord tissue from A)Shh -/- embryos? B) Ptch /- embryos? What if you incubated wild-type notochord with neural plate tissue from Smo -/- embryos? -The active component from the notochord is secreted Shh. In A), there is no Shh so the neural explants will not be ventralized. B) Ptch1 is a gain-of-function mutation in the Shh pathway - but the notochord will still make Shh and so you will get ventral tissue. C) The notochord will still make Shh, but the neural explant tissue can't receive it (Smo is required for signal transduction of the hedgehog pathway). Therefore, you will not get ventralized neuronal tissue (it will be dorsal). 10. You want to misexpress Dbx2 throughout the right side (and only the right side) of a chick neural tube. How can you do this? What type of gene is Dbx2? Would you still form motor neurons? Why or why not? -You need to electroporate a plasmid that expressing Dbx2 into the neural tube (electroporation hits only one side of the neural tube). Dbx2 is a type 1 Shh target that represses the type 2 Shh target Nkx6.1. Misexpressing Dbx2 will cause a loss of Nkx6.1 expression on the right side of the neural tube. Loss of Nkx6.1 will result in a failure to generate motor neurons on that side of the neural tube. 11. You want to further characterize the switch that determines whether trunk neural crest cells will migrate in the early path (becoming peripheral nervous system) or the second, later path (becoming melanocytes). A) Which protein, essential for making all neural crest cells, needs to be downregulated for this to happen? -FoxD3 needs to be downregulated B) You remain unimpressed by the supporting data from lecture (which is all cell culture) and decide to test the model by performing an experiment where you force expression in neural crest cells. How would you do this? How could you determine if this supported the model? -The key is that you need to maintain FoxD3 expression in neural crest cells. If the model is true, all neural crest cells should enter the glial pathway (path 1) or at the very least should not express Mitf1. One strategy would be to electroporate the neural tube at an early stage before neural crest cells migrate with a vector driving FoxD3 expression as well as some other marker (such as GFP). This marker is important because it is unlikely that all cells will get the electroporated construct. This expression would be maintained well after neural crest migration. You would to monitor for a general NCC antibody that is not specific to either lineage (e.g. HNK) as well as for your fluorescent maker (GFP). If the GFP+ cells all migrate through the ventral somites, this suggests they are forming glial cells (could confirm that they do not express Mitf1 with a second immunostaining). If most cells are electroporated, you would predict that you would also have a strong depletion of melanocyte cells. ...
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