Bi 117 Final (60 pts) DUE by 11:00 am on March 15, 2012 Box by Beckman Institute B9 or to a TA Instructor: Marianne Bronner Exam Length: 6 hours plus one 30-minute break at your discretion. It should take about 3 hours but we are giving you double the amount of time. Format: open book, open notes, NO INTERNET access, No collaboration. Directions: Please type your answers for each major section on separate sheets of paper with your name at the top right corner of each sheet. Be concise. Most answers should be a few sentences and maximum of a paragraph. Turn in finished exam into envelope outside Beckman Institute room 9 (BI 9). Answer three out of the four questions. However, you must answer Question 1 and Question 2. You can pick to answer either Question 3 or Question 4. Exam begins once you turn the next page! GOOD LUCK! Total of 60 points Late Policy: - 5% penalty if turned in 11:01 am to noon, 3/15/2012-20 % penalty if turned in 12:01 pm to 5:00 pm, 3/15/2012-40% penalty if turned in 5:01 pm 3/15/2009 to 5:00 pm 3/16/2012 TA s grading your final: Jon Valencia, jev@caltech.edu; Na Hu, nahu@caltech.edu; Benji Uy, uy.benjamin@gmail.com; Jon Schor, jsschor@gmail.com; Stan Schor, stschor@gmail.com
Question I. Neural Crest and Heart Development (20 points) 1. Name 3 different structures/tissues/cell types derived from neural crest. Describe the cell migration pathways involved followed by the precursors to each of these derivatives. (3) (no more than 2 sentences each) 2. One of the most exciting features of the neural crest is its multipotency. a. Briefly define multipotency. (1) (one sentence) b. Knowing this, you would like to test the commitment of early migrating crest cells to a neural crest lineage as opposed to a central nervous system fate. Do you expect the early migrating neural crest cells to be committed to their fate or retain the ability to form CNS derivatives? (one sentence)(1pt) Design an experiment to test your hypothesis. (2) (no more than 3 sentences) 3. You are doing your senior thesis project on a gene that encodes a protein called Bi117 in chick. When knocking down this protein in the right half of the neural tube, prior to neural crest migration, you get a phenotype in which more neural crest cells are detected on the right side of the embryo than the left (unperturbed) side of the embryo. a. Name 2 advantages of using Chick as a model system to study neural crest development as compared to Zebrafish and Frog or mouse. (2) b. You would like to explain where these extra cells came from when Bi117 is knocked down. Your first hypothesis is that the neural crest emigration time period is prolonged on the Bi117 knocked-down side of the embryo. As a consequence, neural crest cells continue to emigrate out of the neural tube on the right side of the embryos whereas they have already ceased emigration on the left side. Design an experiment to show that these extra neural crest cells came from the neural tube. (no more than 3 sentences) (2) c. Your fellow researcher in the lab thinks that there is another explanation for your phenotype and hypothesizes that the extra neural crest cells are due to over-proliferation. As a careful scientist, you would like to test this possibility. Design an experiment and predict the expected result to show if this is correct or not. (no more than 3 sentences)(2)
d. At this point, you have introduced a novel gene that plays a crucial function in neural crest migration. You want to boost its publication to a higher impact journal by identifying a direct target of Bi117. Fortunately, you knew that Bi117 is a transcription factor. Choose which of the following techniques will help you to identify Bi117 s direct downstream target and briefly describe what each technique demonstrates. (3) a) Western Blot b) Immunohistochemistry c) Immunoprecipitation d) Chromatin Immunoprecipitation 4. Below are three pathways followed by prospective mesoderm cells during gastrulation. a. What two signals are required for mesoderm cells to be specified as cardiac cells during chick gastrulation? (1) b. Cells following path 3 normally differentiate as paraxial head mesoderm. Design an experiment to can make them become heart instead. (one sentence) (2) c. Cells following path 1 normally become blood and extra-embryonic mesoderm. Design an experiment to can make them become heart instead. (one sentence) (2)
Question II. Limbs, Bones, Muscles, and Evolution (25 Points) 1. Limb development in chick. a. In the developing chick limb bud, replacement of the ZPA with tissue from the notochord results in a normal limb. What signaling molecule is common to the ZPA and notochord? Design a similar experiment with a cellular/molecular method, testing specifically the possible signaling molecule. (1) b. The difference between the feet of chickens and ducks is the presence of webbing. What type of cellular process is responsible for sculpting the digits? (1) c. You are interested in generating a chicken with feet like a duck. Design two experiments (one molecular and one embryological) in which the resulting morphology of the feet exhibit inter-digital webbing like that of a duck. (2) i) a. Figure. Whole-mount forelimb from 10-12 day chick embryo (Honig 1981). d. From the picture above, explain what experiment resulted in this unusual limb morphology in the developing chicken. (2) 2. In a developing vertebrate, the various populations of neural crest cells will soon differentiate into a plethora of cell types. In the head, some cranial neural crest will form bones of the facial skeleton and parts of the skull. a. What are the two types of ossification and where do they occur? (2) b. Compare and contrast the two types of ossification and the regulatory factors involved? (2)
3. You are studying muscle formation in Ambystoma mexicanum, an amphibian model (e.g. a salamander) and you think you ve found a novel gene in limb muscle development. a. Describe an experiment to determine if this gene correlates with muscle development, in either time or space. (1) b. Describe an experiment to determine if the gene product is necessary for muscle development. (1) c. Describe an experiment to determine if the gene product is sufficient for muscle development. (1) 4. You blast the gene and find out that it is a previously unknown diffusible factor. You put in to fibroblasts and it promotes muscle differentiation. Knowing this, you decide to name it Schwarzeneggerin (Schwar). You place a bead soaked in this new substance into the sclerotome and it turns off Pax1 expression. a. Knowing this fact, what do you expect to see if implant a Schwar bead into the dermotome? myotome? (1) b. What would you expect to see if you knock-down Schwar expression? (1) c. What could you do to rescue the normal phenotype? (1) d. Lastly, you are studying the initiation of Schwar transcription. Assuming that the genes involved in muscle specification and differentiation are conserved between vertebrate models, name three possible factors and describe their roles in inducing regulating Schwar expression. (1) 5. In the developing vertebrate embryo, BMP4 is expressed in many locations and is known to play many crucial roles in early development. BMP4 has been shown to play a key role in bone differentiation, sculpting the autopod of vertebrates and recently in the broadening and deepening of bird beaks. a. What can you infer from this information about the roles of BMP4 in vertebrate development? (1) b. How are new functions introduced into a gene that allows it to be redeployed for novel patterns of expression? (1) c. What is this developmental phenomenon called? (1)
6. You are studying two boxfish populations (Lactoria sp.) that have recently diverged and have therefore undergone slight morphological changes. The long-horned population exhibits a pair of prominent horns on their head whereas the roundbellied population has either reduced horns or entirely absent horns. The difference in this trait maps to Pitx1, a gene known to be involved in bone, thymus, and neuronal development. a. What experiment can you perform to see if this gene correlates with the morphological differences (e.g. horns or no horns) in these species? (1) b. What piece of evidence would suggest that the morphological differences exhibited in the two populations are due to changes in cis-regulatory modules or enhancer regions of the Pitx1 and not in the protein coding region of the gene? (1) c. Design an experiment to demonstrate definitively, by sufficiency, that the difference in horns is due to a change in the regulatory region of the Pitx1 gene. (1) d. Design an experiment to test whether you can recapitulate horn development in the round-bellied population assuming that you can perform transgenesis in these boxfish? (1) e. Draw a detailed, plausible schematic (i.e., conserved regulatory motifs, promoter, and 3 exons) of the Pitx1 locus in the long-horned population, outlining the key difference in the long-horned population. (1)
Question III. Invertebrates (15 points) 1. Explain the importance of having a nanos gradient in the early Drosophila embryo and the importance of the 3 UTR in establishing it. (1) 2. Predict the phenotype of an embryo that lacks a nanos 3 UTR. (1) 3. Explain what posterior dominance means in relation to Hox gene expression. Design an experiment that would support posterior dominance during the process of rhombomere segmentation. (2) 5. You have a C. elegans mutant that lacks intestinal endoderm and has an excess of mesoderm. a. Which gene might be mutated in this embryo? Briefly explain your reasoning. (1.5) b. Other than injecting the embryo with the mrna of the missing gene what could you do to rescue the phenotype? (1.5) 6. You have a C. elegans mutant that lacks hypodermis and has an excess of mesoderm. a. Which gene might be mutated in this embryo? (1) b. Explain why you think this gene is missing. (1) c. Other than injecting the embryo with the mrna of the missing gene, what could you do to rescue the phenotype? (2)
7. A theory in evolutionary development postulates that the insect hexapod (six-legged) body plan evolved from crustacean-like ancestors. a. Could the spatial expression changes of the hypothetical hox genes alone account for the shift to a hexapod body plan or would other changes be required to one or more of these genes as well? Briefly explain your reasoning. (2) b. Design an experiment to determine if spatial expression changes alone are causing the differences in the body plans. (2)
Question IV. Placodes (15 Points) 1. You are walking by the turtle pond and happen upon a few zebrafish. Mysteriously, the fish appear to be swimming sideways. You suspect they were discarded in the pond by a lab on campus. You know Caltech has many zebrafish labs with various tissue specific reporters at your disposal with varying fluorophores. Therefore, you collect the fish and send them to a special zebrafish line identification service. a. Having taken Bi117, you suspect that this unusual swimming behavior might be explained by investigating otic development. You discuss this with a neighboring lab, and the scientists believe that this behavior is due to an increase in hair cells. Perturbation of notch pathway causes excess hair cells in zebrafish. If a scientist decides to remove notch function by removing the trans-membrane and extracellular domain in selected cells will he or she be successful in eliminating notch signal transduction and make excess hair cells? Why or why not? (3pts) b. After further studies, these fish seem to be from a lab that has made a tissue specific construct that is expressed in the otic vesicles, which are misshapen in these fish. These vesicles glow green under the fluorescence microscope. The scientists use a GFP-antibody to isolate and sequence the protein to containing GFP. They find that the GFP is fused to a truncated FGF3 protein. A mrna probe is designed against FGF3 and they perform in-situ hybridization. After developing the in-situ, they notice that the FGF3 mrna is nearly ubiquitous. Why is that? (2pts) 2. Notch-Delta signaling is used throughout development in many invertebrates and vertebrates. a. Name two cases where this type of signaling is used in invertebrates. (2) b. Why is Notch-Delta signaling often used in lateral inhibition rather than as a diffusible morphogen? (1) c. Explain how lateral inhibition works in one of the cases you listed in part a. (1)
3. The trigeminal ganglia has a major nerve that branches into 3 sub-branches. Various environmental queues lead to correct axon guidance of the nerves. For example, the axons express a receptor called Robo-2 which is repelled by slit-1. Robo-2 has a molecular weight of 151 kda. Slit-3 also represses Robo-2. a. Trigeminal neuralgia is an excruciatingly painful syndrome which results in the compression of a branch of the trigeminal ganglia. If a tumor is found pressing against the maxillary nerve in a region where slit-1 is normally expressed, what might the tumor cells be doing (list 2 possibilities)? (2) b. Upon removing the tumor, a biopsy is taken and the cells are placed in tissue culture. Upon extracting the total mrna of the tumor, elevated levels of Robo-2 was found and normal levels of Robo-3 are found. In a western blot of Robo-2, there is a faint single band at 151 kda and a strong band at about 300 kda. Upon treating with beta mercaptimethanol (strong reducing agent/reduces disulfide bonds), a new western shows a strong band at 151 kda. What is a likely explanation? (2) c. Finally, taking the biopsed cells, you discover that they are relatively benign. What markers would you expect to see? (2)