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Unformatted text preview: MCDB 112 (F10) STUDY QUESTIONS – LECTURE SET #3 1. When the sea urchin Primary Mesenchymal Cells (PMCs) ingress, they travel around in the blastocoel and end up adhering to specific locations on the blastocoel wall, where they begin to form spicules. What are they adhering to? Devise a general experimental approach that addresses this question. 2. It is interesting to think about how the PMCs “know” where to go – they actually migrate and set up the spicules in a species ­specific manner: spicules CALIFORNIA (CA) urchin HAWAIIAN (HA) urchin Suppose you can remove ingressed PMCs and transfer them between embryos then watch and see what type (HA or CA) of spicules form. The results that you observe lead you to conclude that PMC localization and spicule structure is AUTONOMOUS to the PMCs (they do not receive signals from other cells in the embryo). Sketch the spicule structures in the embryos that you must have observed in order to draw this conclusion. EXPERIMENT: CA host embryo, HA PMCs HA host embryo, CA PMCs What is the control for this experiment? 3. Antisense knockdowns of macho ­1 result in ascidian embryos that lack tail muscle. However, this phenotype can be “rescued” by injecting wild type mach ­1 mRNA back into these antisense ­treated embryos. Explain why this “rescue experiment” serves as a specificity control for the necessity test. Do you expect the antisense experiment and the rescue to be dose dependent? Why or why not? 4. In general, what are some ways that you could (a) prevent β ­catenin (β ­cat) entry into nuclei of micromeres (and thus prevent them from taking on PMC fates); and (b) cause β ­cat to enter nuclei of all cells in the embryo (and thus create an endo ­meso “ball” embryo)? MCDB 112 (F10) ANSWERS in the ROUGH 1. Think about isolating and fractionating the components of the basal lamina (BL; the ECM of the blastocoel) and doing a version of the binding force experiment – you would place isolated PMCs on plates coated with the various components and see which component(s) served as the best binding substrate (some controls would be proteins from other sources or plates coated with buffer). Alternatively if you already had an idea of what proteins were on the surface of the PMCs or in the BL, you could test of antibodies, when injected into the blastocoel, blocked PMC binding. In this case, you want to think about negative controls and dose dependency. 2. If specific spicule formation is autonomous, then the spicules will always look like those of the PMC donor, regardless of the host species. The control is to take PMCs out of a CA host and put CA PMCs back in (get CA spicules). Same for the HA embryos. You are controlling for the manipulation. 3. The interpretation on the antisense expt is that the antisense if specifically targeting the macho ­1 mRNA and nothing else. However, it is possible that the antisense has an off target or secondary effect that leads to the muscle loss phenotype. By adding back macho ­1, and rescuing the phenotype to wild type, this shows that it is specifically the loss of macho ­1 (and not a secondary “unknown”, which you are not restoring) that is responsible for the muscle phenotype. Both experiments ought to be dose dependent. There is a given amount of endogenous macho ­1 mRNA in the embryo. As you add more antisense, you should expect a more sever phenotype – you need to deplete a given (threshold) amount of endogenous target to cause an effect. Likewise with the rescue – there is antisense in the embryo already – you have to add back enough to overcome any residual antisense and to reach the threshold level for macho ­1 to have its effect on target genes. Note: For rescue experiments, timing can be very tricky. You might have to do the rescue in a very tight time window for it to be effective. These are tough, but elegant experiments and you should appreciate them when you see them! 4a. In theory, you can (i) deplete β ­cat by antisense or inhibit with a function blocking antibody; (ii) over express GSK or a constitutively active, mutant form of GSK (not regulate ­able by DSH) in micromeres, probably best done by injecting mRNA; (iii) deplete DSH in micromeres (by antisense). 4b. In theory, you can (i) over express β ­cat by injecting mRNA – if you swamp out the available GSK, there ought to be excess β ­cat to now enter nuclei; (ii) deplete GSK by antisense or function blocking antibody; (iii) over express DSH in all cells by injecting mRNA. In all cases, need negative controls (scrambled antisense, over expression of irrelevant mRNAs, etc…) and dose dependency. ...
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