E-55, Exam 1-2011: Name

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BIOL E-55 Developmental Biology

Exam 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 blot
B. fate mapping
C. microarray
D. immunohistochemistry
E. 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. specified
B. committed
C. determined
D. differentiated
E. none of the above
__B___3. The process of commitment proceeds in which of the following ways:
A. differentiation > specification > determination
B. specification > determination > differentiation
C. determination > specification > differentiation
D. determination > differentiation > specification
E. specification > differentiation > determination
__D___4. The concept of epigenesis meant that
A. embryos were pre-formed in the germ cells
B. embryos were pre-formed in certain genders gametes
C. embryos were born from eggs
D. embryos were formed from new materials
E. none of the above
__B___5. Which of the following does not require you to know the gene you want to study:
A. gene targeting
B. forward genetic screen
C. RNAi
D. mRNA overexpression using microinjection
E. none of the above
__E___6. Inductive interactions between two cells can involve:
A. secretion of a morphogen from one cell
B. direct signaling between membrane-bound proteins in neighboring cells
C. a receptor of one cell binding a secreted molecule
D. organizer activity by a group of cells in a tissue
E. all of the above are possible

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__C___7. Specialization, or the process by which cells become structurally and functionally
distinct, is known as:
A. specification
B. determination
C. differentiation
D. both A and C
E. both A and B
__C___8. Studying the presence of mRNAs in a sample is useful because
A. it contains introns, which are the protein coding sequences
B. it lacks exons, which do not code for proteins
C. it reflects the genes that are being actively transcribed
D. it reflects the total number of genes in the genome
E. it contains enhancers and promoters
__E___9. Transcription of a gene in the genome is affected by
A. chromatin methylation
B. chromatin acetylation
C. DNA methylation
D. none of the above
E. 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 population
B. it can be used to study chromatin
C. it can be used to detect the presence of specific transcripts in a tissue
D. 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 apoptosis
B. limitless replication potential
C. metastasis
D. loss of both copies of a tumor suppressor gene
E. 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 musclespecific gene, containing the ______ to express ______ in muscle cells.
A. control region, GFP
B. control region, LacZ
C. reporter, LacZ
D. reporter, myosin
E. enhancer, myosin

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__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 from
B. addition, removed from
C. addition, transmitted to
D. removal, transmitted to
E. none of the above
__D___14. Cancers of epithelial origin are called
A. glioma
B. sarcoma
C. leukemia
D. carcinoma
E. lymphoma
__B___15. The most permanent way to fate map a group of cells is
A. performing in situ hybridization to detect transcripts
B. making chick-quail chimeras
C. labeling cells with vital dye
D. labeling cells with fluorescent dye
E. 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

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-inhibition of translation by regulating assembly of the ribosomes

Poly-A tail regulating the stability of the mRNA to make protein
Differential Protein Modification
-modifications to the protein (phosphorylation or cleavage) affect the proteins presence in a cell
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 Wilmuts or Gurdons 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 realdevelopmental 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.

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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.

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).

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20. You are a graduate student in Eric Olsons lab at UT Southwestern, a lab that has an
international reputation for studying cardiac development. You make a mouse knockout 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).
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) _

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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____________________________________
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 cardiacspecific 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.)

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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.
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).

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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.

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 nonmammalian 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).