Solution key- 7.

013 Problem Set 5- 2015

Question 1
You purify a novel noninfectious viral protein (Protein R) and intend to further characterize it.
Accordingly, you develop antibodies against Protein R. You inject Protein R into a rabbit (Rabbit #1),
draw out some blood from the rabbit after a month of injection and confirm the presence of Protein R-
specific antibody in the rabbits blood. You wait for two months and then re-inject Protein R into the
same rabbit. You observe a stronger secondary immune response developing within a few days
against Protein R.

a) From the choices below, circle the immune response that would NOT be generated following
injection with Protein R. Explain why you selected this option.

i. Innate immune response

ii. Humoral immune response
iii. T- cytotoxic cell mediated immune response.
This is not a live antigen and hence will not infect a cell to generate the T C cells mediated immune
response.

b) On the graph below

i. Draw the primary and secondary
immune responses as a solid line (-)
specific to Protein R. Also, draw the
alteration in the concentration of
Protein R as a dashed line (------)
during the primary and secondary
immune responses.

ii. Why is the primary immune response slower and weaker compared to the secondary immune
response?
During the primary immune response, the memory B and T cells, against the specific antigen, are
generated. During the secondary immune response, these can proliferate to produce B cells that
produce and secrete IgG, which has a higher affinity.
c) IgG and IgM are the two classes of antibodies following a primary and / or secondary immune
response to a specific antigen.

i. Which class of antibodies has a higher molecular weight (IgG or IgM)?

ii. Which class of antibodies is prevalent during the secondary immune response (IgG or IgM)?

iii. Which class of antibodies has a higher affinity for antigen (IgG or IgM)?

iv. Name the process(es) that can allow an IgM producing cell to switch and produce IgG.
Class switching

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Question 1 continued
d) You repeat the same experiment as described in the introductory part of the question by injecting
Protein R in another rabbit (Rabbit #2). Would the Protein R- specific antibody repertoire of Rabbit 1
be IDENTICAL to that of Rabbit #2? Why or why not?
NO, each rabbit will randomly generate B and T cells repertoire that will be involved in producing
antibodies that are unique to different parts of the same antigen. There might be some antibodies that
would be common in both but they will not be identical.

e) You isolate the Protein R specific antibody from Rabbit # 2 and inject it into Rabbit #3. You find that
Rabbit #3 produces antibodies that specifically recognize and bind to the antibody from Rabbit #2.
What feature of the immune system allows Rabbit #3 to recognize the antibody from the Rabbit #2 as a
foreign antigen? Very briefly explain why this feature is so crucial for the immune system.
Rabbit #3 will consider the antibodies produced by rabbit #2 as a foreign entity and hence will generate
an immune response specific to it.

f) You are analyzing the immune system of a rabbit, which lacks mature B cells and therefore cannot
produce antibodies against any pathogen.

i. DNA sequence analysis reveals that the immunoglobulin gene of precursor B cells has no
mutation. Give one possible explanation why this rabbit is lacking the mature B cells.
One of the possible answers is: This may be due to mutations in RAG1 and / or RAG2 proteins
encoding genes.

ii. You realize that the immune system of this rabbit can still target and kill the somatic cell that
are infected by a pathogen. Explain why is this so?
This is because the T cells repertoire is still present. The activated TC cells can therefore kill the
somatic cell infected with this antigen.

g) The T cell receptor (TcR) and antibody (Ab) diversity is created via DNA rearrangement of specific
genes. However, other processes further enhance the diversity. List and very briefly describe two such
processes.
You can list and briefly describe any of the following processes: VDJ recombination, junctional
diversity, somatic hypermutation, deaminase activity and TdT activity. Please go through the posted
summary of recitation 9 for description of each.

h) If the antibody H chain gene has 6 V, 4J and 4 D segments and the corresponding L chain gene has
6 V and 4J segments, how many possible combinations of IgG antibodies can be produced from this
gene?
H chains= 6 X 4 X 4 = 96, H chains and L chains = 6 X 4 = 24. Together 24x 96 = 2304

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Question 2
a) You develop a vaccine that can provide protection immunity against a strain of Influenza virus that
has a RNA genome. You use a viral surface protein (i.e. hemagglutinin) as the antigen.

i.Why it is a good idea to use the viral surface protein as an antigen instead of using viral
nucleocapsid proteins?
Antibodies generated against the viral capsid protein will be able to bind to and neutralize the virus
unlike the antibodies against the viral nucleocapsid proteins which is inside the lipid envelop.

ii. In the next flu season, you realize that although the vaccine that you created was very
effective in the last season, it is not effective against the newly generated strain of influenza in
the current season. Why does the virus mutate so rapidly?
The RNA dependent RNA polymerase enzyme that the flue virus uses, lacks the proofreading ability
allowing the virus to mutate frequently. The newly generated strain of the virus, due to mutations is a
new strain for which you may not have antibodies and hence no protective immunity.

b) Your friend uses a live but attenuated (harmless) form of Influenza virus to develop the vaccine.
Explain why your friends strategy is better than yours.
A live virus can infect a cell that has specific cell surface receptors to which the virus can bind. So the
viral particles can be presented by MHC-I molecules on the surface of infected cells but also by MHC-II
molecules on the surface of antigen presenting cells/ dendritic cells. This can generate both the
antibody mediated response as well as the TC cell mediated killing of the infected cell.

c) You are analyzing a person who contracts influenza virus infection. This person has not been
vaccinated and hence does not have prior protective immunity against this virus. You want to determine
the location of functional lymphocytes, which are trying to combat the viral infection in this patient.
Where would these functioning lymphocytes be localized; in the lymph nodes or in the circulation?
Explain why you selected this location.
The lymphocytes function in the lymph nodes.

d) Circle the correct options for each of the following statements.

i. The dendritic cells of the immune system establish crosstalk between the adaptive immune
system and the innate immune system or the mechanical and physical defense barriers.

ii. The complement cascade establishes crosstalk between the adaptive immune system and the
innate immune system or the mechanical and physical defense barriers.

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Question 3
Under resting conditions, the Na+, Ca2+and Cl- concentrations are high outside the neuron and K+
concentration is high inside. This is maintained by the action of specific channels and pumps.

a) Complete the following table for the channels/pumps that establish and maintain the resting
membrane potential.

Channels Ion(s) passing through them & the direction of Movement of ion(s) is
(gated/ open) ion flow (into or out of the cell)? active/ passive? Explain.
pumps
involved
Ion(s) passing? Direction of Active since it requires ATP
+ + its flow?
Na K ATPase
pump
+ Out
Na
in
+
K
Ion(s) passing? Direction of Passive since the flow of
+ its flow? ions is down their
Open/ leaky K
concentration gradient
-, +
and Cl Na K+, Cl-, Na+ Out, In, In
channels

b) The action potential is observed only in neurons. Complete the following table for the
channels/pumps that are involved in generating an action potential.

Phase Channels/pumps Ion(s) passing through Movement of ion(s) is

that are activated them & the direction of active/ passive?
ions flow (into or out of Explain.
the cell)?

Repolarization + Ion(s) Direction Passive since the flow of

Voltage gated K
passing? of Flow? ions is down their
channels concentration gradient
+ Out
K

Depolarization + Ion(s) Direction Passive since the flow of

Voltage gated Na
passing? of flow? ions is down their
channels concentration gradient
+ In
Na

c) What morphological feature of the glial cells allows the impulse to propagate rapidly from the cell
body of the neuron to its axon terminus?
The glia cells wrap around the axonal membrane of a neuron and form the insulated myelin sheaths,
which prevent the leakage of ions. This allows the rapid propagation of action potential along the length
of axon.

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Question 4
The following is a schematic in which the axon (terminus) of a neuron converges onto a muscle thus
creating a neuromuscular junction.
1: On being stimulated, the neuron releases a
neurotransmitter acetylcholine (ACh) that acts as
the ligand.
2: The released ACh binds to the resting (closed)
acetylcholine receptors (AChR) and allows the
AChR to undergo a change in the conformation
from the resting (closed) to an active (open) state.
AchR is a tetramer composed of two
homodimers.
3: The active (open) AChR allow the diffusion of
+
sodium ions (Na ) from the outside to the inside of
the cell, which results in muscle contraction.
4: ACh is eventually degraded by the acetyl
cholinesterase (AChE). As a result, the AChR
AchR reverts back to its resting (closed) state leading to
muscle relaxation.

a) Circle ALL the correct option for each of the following.

i. The ACh signaling pathway is an example of paracrine/ endocrine/ autocrine signaling.

ii. The action of AChR or AChE accounts for the feedback inhibition in ACh signaling.
iii. The conversion of the AChR from the closed to the open state or vice versa represents the disruption of
primary/ secondary/ tertiary/ quaternary level of AChR structure.
iv. The membrane AChR protein sequence contains a signal sequence/transmembrane domain
sequence/both.

b) Acetylcholine is an excitatory neurotransmitter, which causes muscle contraction. Complete the table
below for each neurotoxin.
Taipoxin: Inhibits ACh release into the synaptic cleft (small space between neuron axon and muscle
fibers) by the neuron.
Sarin nerve gas: Inhibits the function of AChE, which is the enzyme responsible for the degradation of
ACh.
-bungarotoxin irreversibly binds to and inactivates the AChR located on muscle cells.

Toxin Results in flaccid (no or slow muscle contraction) or rigid (continual muscle
contraction) paralysis of muscle? Explain your choice.

Taipoxin This will lead to Flaccid paralysis since in the absence of Ach the AChR will never
+
activated, the Na will not diffuse from into the muscle cell and the muscle cell to
cause muscle contraction.

Sarin This will lead to rigid paralysis since in the absence of the functional AChE, Ach will
never be degraded and hence will always be available to activate AChR and cause
+
the diffusion of Na from outside to the inside of muscle to cause muscle contraction.
+
- This will lead to Flaccid paralysis since in the absence of active AChR, the Na will
bungarotoxin not diffuse into the muscle cells to cause muscle contraction.

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Question 5
Serotonin (5-hydroxytryptamine, 5-HT) is an excitatory neurotransmitter. It acts by binding to several
HT receptor subtypes, two of which are shown below.

5-HT2 receptor is a G-protein-coupled receptor, which leads to the opening of Ca2+ ion channels.
5-HT3 receptor is a Na+ channel that opens to allow the diffusion of Na+ ions.

a) Upon binding to serotonin, which activated

Pre-synaptic receptor (5-HT2 or 5-HT3) will generate a faster
neuron action potential in the postsynaptic neuron?
5-HT3 Explain.
receptor 5-HT3, since it is an ionotropic receptor that
directly acts as an ligand channel unlike the 5-HT2
5-HT2 Post- that is metabotropic, which activates the ion
recepto synaptic channel through G proteins coupled receptors.
r neuron

b) Complete the following table for each of the treatments, for synapses where serotonin is secreted by
the presynaptic neuron and the 5-HT2 and 5-HT3 receptors are located on the post- synaptic neuron.
Note: Consider each treatment independently.

Treatment Action potential generated in the post-synaptic neuron is more likely/

Prozac, which inhibits the re-
uptake of serotonin
containing vesicles from the
synapse
Inhibitor of the voltage gated-
Ca2+channels located at the
axon terminus of the pre-
synaptic neuron
less likely/ same compared to untreated synapses? Explain your
choice.
More likely. Serotonin will be available for a longer period to bind to
the receptors located on the surface of the post-synaptic neuron.
Less likely. Pre-synaptic vesicles will fail to bind to the membrane at
the axon terminus and secrete serotonin in the synaptic cleft.

c) GABA is a major inhibitory neurotransmitter in the central nervous system (CNS). It acts by binding
to GABA-A (a Cl- channel) and GABA-B receptors (that activate K+ channels via G proteins). You are
observing the following synapses, between the axon terminus of the presynaptic neuron and the cell
body of the postsynaptic neuron.
Synapse 1: The presynaptic neuron secretes serotonin and the postsynaptic neuron (that secretes
GABA) has 5-HT2 receptors.
Synapse 2: The presynaptic neuron secretes GABA and the postsynaptic neuron (that secretes
serotonin) has GABA-A receptors.

Which of the above two synapses (1 or 2) would be considered an inhibitory synapse? Explain why
you selected this synapse.
GABA secreted by the presynaptic neuron will bind to its receptors on the postsynaptic neuron, which
will open to allow Cl- ions to move into through GABA-A receptors thus hyperpolarizing the membrane
and drifting the membrane potential even further away from threshold level.
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Question 6
The midbrain is an embryonic brain region that

Differentiates to include two cell types, neurons and glia.

These cell types both arise from uncommitted cells, the neuroectoderm, under the influence of the
midbrain organizer, which secretes the ligands Wnt1 and Fgf8.

After induction, on Day 1, all midbrain cells express NeuroD1, a transcription factor that directs formation
of neurons.

By Day 2, a subset of midbrain cells loses NeuroD1 expression and instead expresses, Olig1, another
transcription factor that directs formation of glial cells. At this stage, all midbrain cells are columnar, and
look similar under a microscope.

By Day 6, cells expressing NeuroD1 become neurons, extending dendrites and axons, make synapses
and action potentials; while cells expressing Olig1 become glial cells, which extend a myelin sheath
around the neuronal axons.

Day 0 Day 1 Day 2

midbrain midbrain NeuroD1 NeuroD1 Olig1

organizer
(Fgf8 + Wnt1)

a) From the information above, what is the potency of the following cells? Explain.

i. Midbrain neuroectoderm? Bipotent

ii. NeuroD1-positive cells? Bipotent

They are bipotent since they both forms neuron and glia. If however, you say that the can form multiple
types of neurons or glia cells then pluripotent is acceptable.

b) List the state of the cells on the following days by choosing from committed/ uncommitted/
differentiated) and explain your choice.

Day Cells are committed/ uncommitted/ differentiated. Explain.

Day 1 Committed since the cells show NeuroD1 expression, which is neuronal
regulatory gene.

Day 2 Committed since the cells with NeuroD1 expression are committed to
become neurons unlike cells with Olig1 expression that are committed to
become glia cells.

Day 6 Differentiated since the cells exhibit the structure and functions of neuron
and glia cells.

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Question 6 continued
c) The results of various loss-of-function assays at Day 2 are shown below.

Wild-type

i. Based on the data, circle the region

NeuroD1 Olig1
that has the Wnt1 receptors.
Results in loss of
NeuroD1 and Olig1 Midbrain or Midbrain organizer
expression.
Fgf8 expression is ii. Why do glia (and not neural cells)
Wnt1-/- normal. form near the organizer? Include a
diagram if you like.
Results in
NeuroD1 The organizer secretes Fgf8, which inhibits
expression in neuroD1 resulting in olig1 expression. This
Fgf8-/- or Olig1-/- the midbrain. effect is probably Fgf8 concentration
dependent and will not be seen further from
the organizer.
Leads to
normal Olig1
NeuroD1 -/- expression.

d) The ventral midline of the hindbrain secretes Sonic Hedgehog protein (Shh). This directs formation
of the motor neurons ventrally, near the Shh and interneurons dorsally.

The following table represents different Shh mutants and their corresponding phenotype.

Mutants Genotype Corresponding protein Resulting Phenotype

Wild- type (WT) shh+/shh+ Normal Shh protein Normal motor neurons and interneurons
concentration
#1 (null mutant) shh-/ shh Shh protein absent No motor neurons or interneurons

#2 shh+/shh- Shh protein concentration Motor neurons much fewer and

(heterozygous) half compared to wild-type interneurons more plentiful than wild-
type
#3 (gain- of- shh+/shh+/shh+ Shh protein concentration No interneurons, motor neurons
function mutant) significantly more than throughout hindbrain.
wild-type

Based on the information provided in the table above

i. Explain how the Shh protein differentially regulates motor neuron versus interneuron
development.
Shh is needed for the development of both motor and interneurons as shown by the null mutant and the
wild- type in the table above.It serves as an activator for the development of motor neurons i.e. an
increasing concentration of Shh protein results in motor neuron development as shown by null mutant -
> wild- type -> #2 mutant -> #3 mutant. The secreted Shh seems to establish a concentration gradient
with high concentration ventrally and lowest dorsally. At this low concentration it promotes the
development of interneurons.
i. Would you classify Shh protein as an inducer/ determinant/ morphogen. Circle all that apply
and explain why you circled this option(s).
It is an inducer since it is secreted by the cells of ventral midline, which directs the formation of motor-
and inter- neurons in a concentration dependent manner. So its a morphogen.

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Question 7
The kidney is an organ that is comprised of 10 different cell types and has a specific 3-dimensional
structure. Its formation involves reciprocal interactions between the nephric duct (a tube) and the
metanephric mesenchyme. First, the metanephric mesenchyme induces the duct to form a new tube
called the ureteric bud. The ureteric bud induces the metanephric mesenchyme to form an S-tubule,
which eventually forms the glomerulus, the principal filtration region of the kidney.

Induction Induction

Metanephric
Ureteric S-tubule
Nephric mesenchyme
bud
duct

Kidney formation
a) Briefly explain the concept of reciprocal induction and how it relates to kidney development.
Reciprocal induction is differing tissues causing changes in each other due to signals and receptors in
each. The reciprocal induction between the metanephric mesenchyme and duct is critical for the
formation of glomerulus (the principal filtration region) and ureteric bud that are the key elements of the
urinary system.

b) Formation of the ureteric bud requires branching and lengthening of the existing nephric duct
epithelium. List two processes that can lengthen an epithelium. Briefly explain how cells rearrange
during each process.
Epiboly: The cells of the tube lengthen or stretch out.
Convergent extension: Cells intercalate into the epithelial sheet forming the tube.

c) The metanephric mesenchyme condenses and becomes an epithelial S-tubule.

i. Name the transition associated with this conversion. Mesenchymal to epithelial transition
ii. Of the two cell states (mesenchymal or epithelial), in which state can cells migrate?
iii. Give two changes that would allow the mesenchymal cells to convert to a cell sheet.
They form cell- cell junctions, and acquire apical basal polarity and planer polarity.

d) Predict the effect of each of the following events on ureteric bud formation.
i. Cells are treated with blebbistatin, a small molecule inhibitor of myosin function.
Blebbistatin inhibits myosin function, which is required for contraction of actin filaments and cell shape
change, so the ureteric bud will likely not form as the cells cannot elongate.

ii. Cells express a defective cadherin protein (a protein involved in homotypic cell adhesion) that
lacks its extracellular domain.
Epithelial cells would not show the cadherin dependent homotypic cell cell adhesion required to form
the ureteric bud. The epithelium would be defective and the bud would be small, disorganized or non-
existent.

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