Paper5InClassQ&A

Names of people in your group _____________________________ Lab TA______ Discuss these questions with your group, and write down your answers on this sheet. Turn this in at the end of class for your groups 5 points. We will pick a group from each side to respond to each question. 1. Summarize the information given in Figure 1 B. What do the authors conclude from this data This table shows the identity at the DNA level between the different members (chairy2 related and chairy1-related), and demonstrates that chairy1 and xhairy2 seem to be in a different class from chairy2 and the HES genes. They conclude that chairy2 is an orthologue of HES1, which will be discussed more in the paper. 2. What does Figure 3 show Explain what this data means, and why it is significant. Chairy2 and chairy1 expression is similar but not identical. Chairy 2 is expressed more caudally in the PSM (possibly a more stable mRNA), but most importantly, ends up being expressed in the ANTERIOR of each newly formed somite rather than the posterior, as is chairy1. Perhaps this indicates that expression of both is defining the size of the somite (the paper doesnt really discuss the implications, but I think this is really interesting). 3. What evidence do the authors present that HES1 does not control the segmentation clock (you should be looking at Figures 7 and 8). They looked at members of the Notch signaling pathway (Dll (delta) and Lfringe (lunatic fringe), both thought to be involved in the segmentation process. They showed that lfringe expression continues to cycle in a normal pattern even in HES1 heterozygous and homozygous mutant mice (Fig 7). On the other hand, if they looked at mice who were null for Delta (Fig 8), HES1 was no longer expressed. This suggests that HES1 must be downstream of the N/Dl, lfringe pathway, rather than the other way around. One thing that is weird is that they show an absence of somites in the Dll mutant--I thought that Dll mutants still made somites, they were just disorganized (evidence for N/Dl being involved in the separation or boundary formation of the somites from the PSM). 4. Propose a pathway of how the Notch, lfringe and the c-hairys (or HES1) interact together to drive the segmentation clock (see last page of the paper). Some feedback must be involved here, but the order is not yet clear. Notch, HES1 and the hairys expression is not affected by lack of protein synthesis. Lunatic fringes expression is dependent upon protein synthesis, but lunatic fringe cannot be downstream of the hairys (as shown in this paper). Something must be regulatingN/Dl, which then regulates lunatic fringe level (or maybe the other way around), which then lead to oscillations of the hairys in the PSM. But we dont know what it is regulating N/Dl. Questions for Team WEST Paper Discussion 5 MCDB 4650 Names of people in your group ______________________________ Lab TA______ Discuss these questions with your group, and write down your answers on this sheet. Turn this in at the end of class for your groups 5 points. We will pick a group from each side to respond to each question. 1. Describe how the complete c-hairy2 was isolated, and the significance of what is shown in Figure 1A. c-hairy 2 was isolated by using a piece of c-hairy 1 to probe a cDNA library. They should include something about what a cDNA library actually is. They didnt get the 5 end of the c-hairy2 gene (presumably, no start site in the clones they isolated), so they did PCR with primers from the 5 end of the sequence they had and the 5 end of the highly conserved HES1 sequence. This allowed them to get the complete sequence. The rest of Figure 1A shows similarities between the various family membersthe authors make the point that chairy1 and xhairy2 are likely to be orthologues, and differ from the other members because of a 20 aa insertion near the N term end. 2. What does Figure 2 show Does the data presented explain how they know that the expression is cyclical If not, what does demonstrate its cyclical expression The figure shows hybridization of 17-somite stage embryos with chairy2. Many embryos were observed, and these 4 patterns were seen, suggesting that the chairy2 expression is cyclical, as is chairy1. This collection of embryos does not actually prove that the expression cycles in a 90 minute time periodthats described in the text, but no data is shown. That technique is to cut the PSM in half and let one half survive in culture for periods of time up to 90 minutes, while the other half is fixed and stained immediately. Using THAT technique, they knew the expression was on a 90 minute cycle. 3. The authors show in Figures 4 and 5 that HES1 expression cycles in mouse embryo PSM just as the c-hairys do in chick PSM. Which of the two c-hairys does HES1 expression look most similar to (you might also look at Figure 6 for additional data) Why is this interesting It looks most similar to chairy1in fact it looks almost identical. Interesting because by sequence analysis, HES1 and c-hairy2 are orthologues, not HES1 and chairy1. The authors suggest that there might be only one cycling HES member in mouse, rather than 2, since no one has pulled out another one. 4. Summarize the authors evidence that Notch could be a core stimulator of the segmentation clock, and how Notch intersects with activation of the c-hairys (and/or HES1 ). The authors suggest that the hairys are outputs of the clock, a readout. Mutations in Notch change the expression pattern of both hairys and lfringe, suggesting that Notch is an important component. Notch, when activated by binding to Delta, is proteolytically cleaved and translocated to the nucleus where it acts as a trnasription factor. This is possibly how hairys and lfringe are activatedby Notch itself.