Fluent Essays

VIEW ATTACHMENT -------------------------------------------------------------------------------------------------------------------------------------------------- INSTRUCTIONS: Your responses will be graded based on the correctness, appropriateness, and thoroughness of your answers. You are expected to support your answers with appropriate observations, experimental evidence, and/or methodologies. You will lose credit if what you write is wrong. You will receive no credit if what you write is true but does not answer the question. You may not receive all the credit the question is worth if you leave out what I consider to be important key points. Many questions have multiple parts. Be sure to check before handing in your exam that you have completed ALL of the questions. devbiopart2.docx Unformatted Attachment Preview INSTRUCTIONS: Your responses will be graded based on the correctness, appropriateness, and thoroughness of your answers. You are expected to support your answers with appropriate observations, experimental evidence, and/or methodologies. You will lose credit if what you write is wrong. You will receive no credit if what you write is true but does not answer the question. You may not receive all the credit the question is worth if you leave out what I consider to be important key points. Many questions have multiple parts. Be sure to check before handing in your exam that you have completed ALL of the questions. Please write your answers in a different color—anything but black (INSTRUCTIONS/INFORMATION) and blue (QUESTIONS) that is easily readable. -------------------------------------------------------------------------------------------------------------------------------------1. (3pts) If you make a conditional knockout of FGF8 in the somitic and lateral plate mesoderm of the mouse at the onset of limb bud formation (AFTER somites have already formed), meaning that FGF8 is only depleted in these tissues, describe the limb phenotypes you expect to observe. Explain your answer. 2. (3pts) What if you did the same experiment but this time conditionally removed FGF10 from the somatic and lateral plate mesoderm of the mouse? Explain your answer. 3. (3pts) What would you expect to happen if you inhibited FGF8 within the presomitic mesoderm BEFORE somite formation? Describe the expected phenotypes and why they would arise. 4. (3pts) If you make a conditional knockout of Shh in the somitic and lateral plate mesoderm of the mouse at the onset of bud formation, describe the limb phenotypes you expect to observe. Explain your answer. 5. Carefully look through the figure below (I’ve changed the name of the gene again…) and read the accompanying figure legend. Use this information to answer the following questions. (A) Diagram of sequential gene expression as a mesodermal cell progresses from a progenitor state in the dorsal tailbud until incorporation into a somite. (B-B″) Double fluorescent RNA in situ hybridization for monster (red) and ntl (green) in a wt zebrafish embryo. (C-J′) Expression of ntl (C-H′) and wnt8 (E-J′) in monster morpholino-injected embryos (inhibiting translation of Monster protein) (D,D′,F,F′) and their siblings (C,C′,E,E′) and in embryos overexpressing monster (H,H′,J,J′) and their siblings (G,G′,I,I′). At the 8- to 10-somite stage (H), 55\% of monster-overexpressing embryos showed an absence of notochord ntl staining and 33\% presented notochord breaks, but some expression of ntl was still evident in the tailbud. At the 20-somite stage (H′), 88\% of these embryos showed an absence of ntl staining in the tailbud; this 88\% consisted of 66\% that showed no notochordal ntl expression and 22\% that showed some notochord staining. Seventy per cent of monster over expressing embryos showed continuing but strongly downregulated wnt8 staining at the 8- to 10-somite stage (J); the remaining 30\% exhibited a total absence of wnt8 expression. By the 20-somite stage (J′), expression of wnt8 had disappeared completely. a) (2pts). Which of the critical types of experiments (find it, lose it, move it) are done in this figure? b) (5pts). Explain in your own words the relevance of the data presented in panels C-J’. What do these experiments tell the scientists about Monster? Be clear about your interpretation of how Monster likely functions in this tissue. c) (3pts) Considering the data shown of the gene expression phenotypes associated with Monster inhibition through morpholino injection (panels D and F) what defects do you hypothesize these fish will have in somite formation? 6. (12pts) Considering the phenotype shown below, generate THREE distinct hypotheses about what gene (or type of gene) is being affected in this patient (meaning I want three different genes). Record the gene (or type of gene) and the nature of the mutation (loss-of-function, gain-of-function, a type of regulatory mutation (be specific as to the type of regulatory mutation), etc.). Explain WHY you think mutations in these genes would lead to the defect shown. Base your answers off of data from model organisms that exhibit similar wild type phenotypes as humans. Think critically about the validity of your answers based on known data. 7. Read and analyze the following figure/figure legend: wild type bdd/bdd Figure Legend. The panels to the left represent cross-sections through the neural tube of wild type (A,C,E, and G) and brain differentiation defective (bdd) mutants (B,D,F, and H). Immunofluorescence (shown in red) against Pax7 (a dorsal neuronal marker – A,B), Pax6 ( a medial neuronal marker – C,D), Nkx2.2 (a ventral neuronal marker – E,F), and Hnf3β (A ventral neuronal marker – G,H). *Note: I invented the name of this mutant. You will not find it in the literature. a) (1pt) Is red in the above images denoting RNA or protein expression? Highlight and explain your answer. b) (5pts) Propose a signaling pathway that is likely affected in the bdd mutant. Defend your answer. 8. You recently performed a mutagenesis screen to isolate mutants that exhibit left-right patterning defects in the zebrafish embryo. You screened for proper heart looping. You uncovered two interesting mutants that showed defects in heart looping. One of the mutants displayed defects in Nodal signaling (rightward bound - rbd), while the second mutant maintained correct expression of Nodal signaling (flipped – flp). a) (4pts) To learn more about how rbd and flp might be acting in left-right patterning, you decide to look at the expression of each gene. You start by doing RNA in situ hybridization. Hypothesize where (in what cells/tissues) you expect to see expression of rbd (an educated guess). Justify your answer. b) (6pts) You only scored heart looping during the original mutagenesis screen, so you plan on performing a secondary screen through your mutant collection looking for defects in liver and pancreas positioning. Do you expect to see defects in liver and pancreas positioning in rbd and flp mutants. Support your predictions for each. 9. (4pts) From this same mutagenesis screen, you identified a mutant that showed defects in heart valve morphology. Upon closer inspection, you realize that this mutant also shows defects in bone formation, in specification of some neuronal populations and some populations of melanocytes. At first, you can’t wrap your brain around how this mutation can affect such a diverse population of tissue types, but then your excellent training in developmental biology kicks in, and you come up with a hypothesis! You hypothesize that this gene functions in/to: (Justify/support your hypothesis). 10. It is the year 1992 and you are a graduate student in a lab studying limb development. The vertebrate homolog to the Drosophila gene Hedgehog was just discovered and named SHH (Sonic Hedgehog). You have developed a method in which you can add SHH to a bead and express SHH in ectopic positions to better study its function. Answer the following questions: Experiment 1: A SHH bead was placed in the following location (see picture) of the neural tube just after the formation of the medial hingepoint (MHP). a) (2pts) During MHP formation, what tissue normally expresses SHH? SHH Bead b) (4pts) What would you expect to be the consequence to neural tube development of placing the SHH bead in this location? Justify your answer. Experiment 2: A SHH bead was placed in the anterior position of the limb bud (see picture). c) (2pts) What are the anterior and posterior structures normally found on a human hand? d) (4pts) Describe the predicted effect on the limb if a SHH bead was placed at the anterior end of the forelimb bud as diagrammed here. Your friend is a graduate student in an adjacent lab studying somitogenesis. She discovers a mutation in a mouse embryo that looks similar to the phenotypes observed in somitogenesis when FGF is overexpressed. e) (3pts) Describe what your colleague observed—what defects would she have seen in somitogenesis? f) (3pts) You suspect the mutation might be in the Retinoic Acid (RA) Signaling pathway. If this mutation affects the RA signaling pathway, do you think this mutation would inhibit or promote RA signaling. Explain your answer. 11. (2pts) If you were able increase the rate of the oscillating gene expression, do you think the number of somites would increase or decrease? 12. You’re now a postdoc doing research in a lab that studies germ cell development in Drosophila. The entire lab joins together to do two separate mutagenesis screens—a maternal effect screen and a zygotic screen. Different members of the lab are interested in understanding more about discrete aspects of germ cell development: Grad student 1 (we’ll call her Kari) cares about initial formation of pole cells. Grad student 2 (we’ll name him James) is interested in germ cell migration. You care about niche formation in the male gonad. Your lab isolates mutants that affect each of these three processes. a) (3pts) Which of the two screens (maternal effect versus zygotic) would each of the three individuals have done. Shh SHH b) (3pts) Kari’s screen isolated a mutant in which germ plasm completely fails to form. She determines that this is a mutation in a gene that is already known to be involved in germ cell development. Based on what we learned in class, what gene do you hypothesize she might have identified a new mutation in? Explain your answer c) (6pts) Kari’s screen also isolates two additional mutations in two separate genes that have the same phenotype—germ plasm forms appropriately at the posterior but is unevenly distributed in the resulting pole cells. In fact, some pole cells completely lack germ plasm and will consequently fail to differentiate as germ cells. What TWO DIFFERENT genes might Kari have identified in her screen and why would their loss give rise to this phenotype? d) (4pts). James has identified two mutations that both cause germ cells to migrate inappropriately and fail to properly associate with the somatic gonad. He wants to know if these mutations are in the same gene or if he has identified two distinct factors that both contribute to germ cell migration. What experiment would he do to determine this? Briefly describe how this experiment would be done. e) (6pts) James has also identified another mutation that causes germ cells to remain within the gut and completely fail to migrate to the somatic gonad. What are TWO different processes that might be disrupted in these mutants. For each process please specify in what cell type the mutated gene is required to function. f) (4pts) Finally, you identified a fascinating mutant! In these mutant embryos, you find that the germ cells and somatic cells coalesce appropriately into a gonad but that in the male, a niche consistently fails to form. What gene do you hypothesize may be mutated in these embryos? Explain your answer g) (3pts) Do you think the mutants from part f) would be fertile when adults? Why or why not. ... Purchase answer to see full attachment