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BIOL E-55 Developmental BiologyExam 1 2011

A. You may use your book or the internet to research answers.

It is expected that you paraphrase answers in your own words or reference the source.

B. It is expected that you work INDIVIDUALLY on the exam.

Please do NOT discuss answers with other students or individuals.

C. Be sure to write your name on EACH PAGE of the examination.

D. Read questions carefully and write each answer clearly.

If your answer cannot be read, it cannot be graded.

E. For the short answer section, it is important to show your reasoning in order to be awarded partial credit. Be concise and limit your response to what is specifically being asked in the question.

F. The point value of each question is noted.

G. Answers should be written in ink (black or blue pen is accepted) or typed.

IF YOU WRITE IN PENCIL YOU CANNOT ASK FOR A REGRADE

H. Questions about the exam should be submitted by email.

Questions emailed to me after 6pm on Tuesday, March 1, 2011 will NOT be answered!

Multiple choice 1-15 (45 points)Questions:16 (12 points)17 (3 points)18 (9 points)19 (3 points)20 (16 points)21 (12 points)

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Multiple Choice (3 points each). Select the best answer from the choices provided.

___C__1. Which technique provides information about mRNA in a tissue sample?A. Southern blotB. fate mappingC. microarrayD. immunohistochemistryE. gene targeting

___E__2. Fate mapping in a frog species shows that cells in a specific region of a blastula stage embryo always give rise to the blood cells. Based on this information, these cells in the blastula embryo are best described as:

A. specifiedB. committedC. determinedD. differentiatedE. none of the above

__B___3. The process of commitment proceeds in which of the following ways:A. differentiation —> specification —> determinationB. specification —> determination —> differentiationC. determination —> specification —> differentiationD. determination —> differentiation —> specificationE. specification —> differentiation —> determination

__D___4. The concept of epigenesis meant thatA. embryos were pre-formed in the germ cellsB. embryos were pre-formed in certain genders’ gametesC. embryos were born from eggsD. embryos were formed from new materialsE. none of the above

__B___5. Which of the following does not require you to know the gene you want to study:A. gene targetingB. forward genetic screenC. RNAiD. mRNA overexpression using microinjectionE. none of the above

__E___6. Inductive interactions between two cells can involve:A. secretion of a morphogen from one cellB. direct signaling between membrane-bound proteins in neighboring cellsC. a receptor of one cell binding a secreted molecule D. organizer activity by a group of cells in a tissueE. all of the above are possible

__C___7. Specialization, or the process by which cells become structurally and functionally distinct, is known as:

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A. specificationB. determinationC. differentiationD. both A and CE. both A and B

__C___8. Studying the presence of mRNAs in a sample is useful becauseA. it contains introns, which are the protein coding sequencesB. it lacks exons, which do not code for proteinsC. it reflects the genes that are being actively transcribedD. it reflects the total number of genes in the genomeE. it contains enhancers and promoters

__E___9. Transcription of a gene in the genome is affected byA. chromatin methylationB. chromatin acetylation C. DNA methylationD. none of the aboveE. all of the above

__C___10. The reverse transcriptase-polymerase chain reaction (RT-PCR) is useful because:A. it can be used to measure protein levels in a cell populationB. it can be used to study chromatinC. it can be used to detect the presence of specific transcripts in a tissueD. it can be used to visualize distribution of transcripts in a tissue section E. none of the above

__E___11. Something that is NOT characteristic of cancer:A. evading apoptosisB. limitless replication potentialC. metastasisD. loss of both copies of a tumor suppressor geneE. none of the above

__A___12. You wish to visualize muscle formation in live zebrafish embryos so you decide to make a transgenic fish. To do this, you use a piece of DNA 5’ to the start of a muscle-specific gene, containing the ______ to express ______ in muscle cells.

A. control region, GFPB. control region, LacZC. reporter, LacZD. reporter, myosinE. enhancer, myosin

__C___13. Transgenic animals are best described as the result of the ______of a piece of DNA that is ______the next generation.

A. removal, removed fromB. addition, removed from

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C. addition, transmitted toD. removal, transmitted toE. none of the above

__D___14. Cancers of epithelial origin are calledA. gliomaB. sarcomaC. leukemiaD. carcinomaE. lymphoma

__B___15. The most permanent way to fate map a group of cells isA. performing in situ hybridization to detect transcriptsB. making chick-quail chimerasC. labeling cells with vital dyeD. labeling cells with fluorescent dyeE. injecting RNAi

Short answer (point values indicated). Provide a response in the space provided.

16a. (6 points) Describe 3 different mechanisms that can explain differential gene expression in a cell (there are many but only describe 3!).

2 points (up to 6 points total) for describing any of the following mechanisms:

Differential gene transcription: -control regions regulating which genes are or are not made into RNA-chromatin organization (acetylation or methylation) regulating access to RNA polymerase-transcription factors regulating gene transcription-DNA methylation regulating gene transcription

Selective nuclear RNA processing:-differential splicing regulating which exons are used to make protein-differential nuclear export to the cytoplasm regulating if the RNA will make protein

Selective mRNA translation:-microRNA-mediated mRNA degradation or cleavage interferes with protein translation-inhibition of translation by regulating assembly of the ribosomesPoly-A tail regulating the stability of the mRNA to make protein

Differential Protein Modification-modifications to the protein (phosphorylation or cleavage) affect the protein’s presence in a cell

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16b. (6 points) Describe 3 mechanisms that result in differential gene expression in a group of cells or embryo. In other words, what mechanisms can regionalize an embryo?

2 points (up to 6 points total) for describing any of the following mechanisms:

Cytoplasmic determinants (autonomous specification) such as mRNAs or proteins localize to different part of an embryo and specify cell fates.

Induction (can be conditional specification) involves interactions between cells. For example, a secreted molecule from one cell induces a response or change in gene expression in a neighboring cell.

Organizers can be groups of cells capable of influencing changes in a larger tissue.

Morphogens released from one set of cells cause concentration-dependent changes in responding cells

17. (3 points) What is the experimental evidence for genomic equivalence?

1 point for describing either Wilmut’s or Gurdon’s experimental design.1 point for explaining that it was a differentiated nucleus that is transplanted (into an enucleated egg)1 point for explaining that this nucleus made a whole organism, showing that no genetic material is lost/differentiated cells contain all the genetic material needed to make an organism.

18. To monitor how the pancreas forms from the endoderm precursors, you decide to use a transgenic reporter strategy in zebrafish. You isolate a 1.5 kilobase (kb) fragment of genomic DNA that is located directly upstream of the first exon encoding a novel gene that is highly and specifically expressed in all endoderm precursors.

A. (3 points) What reporter gene would let you visualize pancreas formation in real-developmental time? Explain why.

GFP or Green Fluorescent protein (1.5 points)The advantage of GFP is that it can be used for following developmental processes in live embryos (1.5 points) unlike LacZ staining which requires fixing and staining the embryo.

B. (3 points) A member of your lab has recently found a zebrafish protein that emits a fluorescent yellow color when it is expressed in cells. It is a gene present in the genome of the zebrafish. Would this be useful as a reporter gene in zebrafish? Explain why/why not.

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No (1.5 points)A reporter gene should not be endogenously expressed in the organism you are studying. Expression of the yellow protein would interfere with analysis of your reporter (1.5 points).

C. (3 points) When you make your transgenic model, you are surprised to find ectopic expression of the reporter in the zebrafish muscle (a mesoderm derivative). In addition, while most of the pancreas has reporter expression, a region is not labeled by the reporter. Provide and explain a reasonable hypothesis to account for the ectopic misexpression of the reporter and the missing expression.

This question was referencing the modularity of enhancers (Gilbert p41-42). The expression of the reporter in your transgenic model is best explained by the presence or absence of enhancer elements in your original construct that are causing improper expression in the tissues mentioned. This means that the 1.5 kb piece that you used does not contain all the necessary regulatory enhancers required for proper expression.

For example, your 1.5 kb region could be missing enhancers that are necessary for expression specifically in the pancreatic region that is not labeled. In addition, you could be missing enhancer regions that are important for turning off expression in the muscle (silencers).

An alternative accepted answer: This effect could also be caused by insertion effect- that is the site of DNA integration of your reporter construct could affect its expression in your transgenic line.

3 points for fully describing any of these scenarios.

19. (3 points) Give the name and explain 2 types of genetic changes that can cause cancer.

1. Gain-of-function mutation resulting in an oncogene (dominant mutations). Oncogenes can cause excessive proliferation and growth of a cancer (1.5 points).

2. Loss-of-function mutation in 2 copies of a tumor suppressor gene (recessive mutations). Tumor suppressor genes usually negatively regulate the cell cycle, and loss of both copies can result in uncontrolled proliferation (1.5 points).

20. You are a graduate student in Eric Olson’s lab at UT Southwestern, a lab that has an international reputation for studying cardiac development. You make a mouse knock-out model of a transcription factor that is expressed in the heart and are very excited at the prospect of contributing to the long-standing lab legacy with your research. Unfortunately, your homozygous knock-out mice display normal development and become healthy adults with normal cardiac function (so their heart works).

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A. (4 points) Provide 2 possible explanations for this result:

2 points (up to 4 total) for providing the following explanations:

This gene is simply not required for heart development.

Redundancy: similar family members to this gene are also expressed in the heart and can compensate for its function.

I will also accept conditional specification as a possible explanation. Loss of some cardiac cells that require this factor are replaced by other cells that are able to compensate and create a normal heart.

Despite your previous knockout experience, you persevere with your Ph.D. and decide to knock-out another novel transcription factor that is also highly expressed in the heart. In a desire to graduate quickly, you rush to make a mouse knockout without any further experiments, and find that your homozygote knock-out mice die during early gestation.

B. (2 points) What is the most likely explanation for this result?

Pleiotropy is the most likely explanation. Your gene is required at early stages during mouse development, possibly in other tissues, causing early lethality (2 points).

You realize that you should you have employed some other techniques to find out more about the expression of the transcription factor during embryogenesis.

C. (2 points) Name two techniques that would have told you information about expression of the gene.

(1 point up to 2 points total for any of the following)

1. ____ in situ hybridization (mRNA expression) ____

2. ____ RT-PCR (mRNA expression)____________

3. ____ immunohistochemistry (protein expression) ___

4. ____ microarray (mRNA expression) __________

5. ____ enhancer with reporter (surrogate expression) _

D. (2 points) Given the embryonic lethality of the knockout, what technique will you now use to knockout the transcription factor only in the developing heart? (Hint: You have numerous heart-specific promoters at your disposal in the Olson lab.)

__________Cre-Lox____________________________________

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E. (6 points) How does this technique interfere only with heart development? Clearly describe AND ALSO draw simple diagrams to illustrate the DNA constructs that you will use to engineer the system you listed in part D.

The Cre-lox system allows for spatial and temporal gene inactivation (2 points total).A tissue specific promoter drives expression of Cre recombinase. In this question, it must be a cardiac-specific promoter (1 point). Your gene should be surrounded by loxP sites to allow for recombination and excision of your gene in a specific tissue. (3 points for a drawing resembling this.)

21. You are interested in how the liver forms in a newly discovered vertebrate similar to a zebrafish embryo. You decide to perform a fate-mapping experiment.

A. (3 points) Describe how you would perform this fate-mapping experiment.

A labeling technique should be described (1.5 points). This could include a dye such as a fluorescent dye or a chimera experiment using GFP-labeled donor cells from a zebrafish.

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The procedure for performing the fate map should also be described (1.5 points). To do a fate map, cells or regions of cells are labeled at an early stage and followed to later stages to determine which structures they contribute to.

B. (3 points) You successfully construct a fate map of a group of cells at the blastula stage that always give rise to the liver. You conclude these cells to be the liver progenitors. From the fate map data, can you infer whether these cells in the blastula were either specified or determined to become a liver? In your answer, provide clear reasoning as to whether the term specified or determined could or could not be used to describe the liver progenitor.

No, you cannot infer the stage of commitment of a cell from a fate map. You can only conclude that those cells are ‘fated’ to give rise to that structure (1 point).Use of the terms, specified and determined, requires an operational test of the cells.The cells would be ‘specified’ if upon transplantation to a neutral environment, they differentiate into liver cells (1 point).The cells would be ‘determined’ if upon transplantation to a non-neutral environment, they differentiate into liver cells (1 point).

You surgically remove the liver progenitors at the early blastula stage, expecting that a liver-less embryo will develop. However, you are surprised to find that all embryos with this surgery develop a normal liver.

C. (3 points) What does this experiment suggest about the mechanism of development that is operative with regard to the liver? In your answer, clearly define the difference between autonomous and conditional specification and how they relate to the mechanism of development.

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The mechanism of development is conditional specification (1 point).In conditional specification, the fate of the cells depends upon interactions between cells. Cells lost or removed can be replaced by neighboring cells (1 point).Autonomous specification occurs through the inheritance of determinants that influence the fate of the cell. Cells will develop according to their fates even if removed from the embryo (1 point)

Next, to learn more about the liver progenitors, you make a cDNA library from the embryo so that you can search for gene transcripts that are unique to the liver progenitor cells. You perform a whole mount in situ hybridization screen using the cDNA collection and find several transcripts that are specific to just the liver progenitors. One cDNA is particularly intriguing to you because it looks like a gene that has not been discovered in other species yet, so you decide to study it further.

D. (3 points) What technology could you use to test if your chosen liver progenitor gene is necessary for liver formation? Assume that all technologies that work in non-mammalian models can be successfully performed in your critter. In your answer, clearly state one possible phenotype you would predict to see if your gene is necessary, and one possible phenotype if your gene is not necessary.

You will want to knock-down gene function using antisense RNA, morpholinos, or siRNA, injected into early stage embryos and assay phenotypes at later stages (1 point)

If your gene is necessary, you would expect an incomplete or no liver to form (1 point).If your gene is not necessary, you would expect a normally developed liver (1 point).