Block II study guide: includes

Terms and lecture objectives

Lecture XVII
In tr odu ctio n to Par t II - Presentation of a Cas e Stu dy

Terminology
Term
Hereditary Hemorrhagic
Telangiectasia (HHT)
Anemia
Focal Vascular Lesion
Epistaxis
Telangiectasias
Arteriovenous
Malformation
Nidus
Autosomal Dominant
Haploinsufficiency
Incomplete Penetrance
Variable Expressivity

Definition
Genetic (autosomal dominant) disorder that leads to abnormal blood vessel
formation in the skin, mucous membranes, and often in organs such as the
lungs and brain. This can cause nosebleeds, GI bleeding.
Common blood disorder attributed to a general decrease in number of
erythrocytes or decrease in hemoglobin.
Abnormality near the surface of epidermal tissue attributed from vascular
origins, particularly through dilation of blood vessels.
Common occurrence of nosebleeds, where blood drains from the nostril,
attributed to rupture of the blood vessels in the nose.
Small, dilated blood vessels near the surface of the skin or mucous
membranes.
Abnormal connection between veins and arteries, typically from congenital
origins.
Large, fragile tangle/accumulation of arteries and veins.
This refers to the inheritance pattern of a disease. If it is autosomal
dominant, then only one abnormal gene from a parent is necessary to attain
the disorder.
When an organism only has one functional copy of the gene, with the other
inactivated, which can cause abnormal presentations such as in diseasestates.
Proportion of patients carrying the genotype that do not express the
phenotype.
Variations in phenotype carrying a particular genotype. It is analogous to the
severity of a condition in clinical medicine.

Lecture Objectives
Introduction to Block II and the case study.
Block II surrounds the elements of signal transduction pathways that alter gene expression. This strategy
is mainly the way to target genes. This can occur in elements of development and can be utilized to our
advantage in stem cell research. In short, signal affects expression.
The case study involved in this block is the medically unique tale of a patient named Sarah. She is a 32year old martial arts instructor in South Arizona, married with kids. She has experienced elements of
fatigue (tired and sluggish). She initially attributed it to stress until she observes blood in her stool.
Sarahs doctor does a history and physical, with no indication of colon cancer in the family. A fecal occult
blood test confirms blood in her stool, and Sarah is then scheduled for a colonoscopy. Her doctor finds
numerous small red spots on her skin, distributed mostly in her mouth and on the top of her hands. They
increased in number, as she got older. She also gets small lesions on the inside of her mouth or tongue
from time to time. Sarah also tells her doctor she had a history of nosebleeds, even though her sister does
not get nosebleeds. Her mom also had a history of nose bleeds, and that one of her sons had a lot of
nosebleeds. The colonoscopy revealed vascular lesions (known as telangiectasias) in her colon, and
laboratory test revealed anemia in her blood. This led to the diagnosis of hereditary hemorrhagic
telangiectasia, and DNA testing later revealed that she has specifically HHT Type II.
Learn about the presentation, diagnosis, complications, and inheritance pattern of HHT.
Hereditary hemorrhagic telangiectasia (HHT) is a vascular disorder found in ~1 in 5-8000 people, with 1.2
million people affected. It is a multisystem vascular disorder consisting of focal vascular lesions
(telangiectasias or arteriovenous malformations). It is an autosomal dominant disease, with the bad
gene inhibits the good gene, and so the bad protein is created so that it inhibits the desired effects from
normal proteins. It exhibits haploinsufficiency in which the on good copy is not enough for normal effects.
The disease has marked intrafamilial variation in terms of both penetrance and variable expressivity.
The presentation is typically observed in childhood but the symptoms can be aggravated with age, with
patients exhibiting only nosebleeds and telangiectasia. Of the patient population, approximately a third of
the patients exhibit chronic anemia and GI bleeding. However, a majority can have typically silent

arteriovenous malformations in the pulmonary, hepatic, cerebral, and spinal circulations. Complications
can involve not only anemia due to chronic GI and epistaxis, but also in the larger arteriovenous
malformations in lungs, brain, liver, and GI tract (such as in stroke, hemorrhage, and brain infection). More
rare complications tend to include congestive high output heart failure from the decrease in capillary bed
size.
Diagnosis involves three of the following criteria: (1) spontaneous and recurrent epistaxis, (2) multiple
telangiectasias at characteristic sites, (3) visceral vascular lesions (gastrointestinal telangiectasias and
proven arterioveous malformations on lung, liver, brain, or spine), (4) family history of HHT (first-degree
relative)
Treatment and management involves strategies to: (1) stop or prevent excessive bleeding, (2) increase
clotting ability, (3) replenish lost blood, (4) monitor for more serious complications, and (5) procedures to
block off local sites where there is a blood vessel defect.
Know the underlying structural disorder in HHT. Distinguish between Telangiectasias and
Arteriovenous Malformations.
The underlying disorder in HHT is attributed to the abnormal vascular architecture. There are two types of
vascular abnormalities: telangiectasis and arteriovenous malformations (AVMs). Telangiectasis are focal
dilatations of microvessels. There is a decrease in the number of capillaries, and are convoluted in larger
lesions in later stages. This implies that there is really a direct connect between arteries and veins, and
thus does not allow appropriate capillary exchange. They are common on skin and mucous membranes.
Arteriovenous malformations are essentially direct connections between larger arteries and veins. These
are typically larger than telangiectasis, up to several centimeters. At this point , there is a decrease (if not
a lack) of capillaries, and can be present as a nidus (essentially a big, fragile tangle of arteries and veins).
AVMs can particular develop in distinct parts of the vasculature to limit bypasses in normal circulation.
Think about what we might learn by examining this case.
From observing the pathogenic mechanisms underlying the vascular malformations in HHT, we can learn
about how the genetic mutations can overall affect signaling patterns in the body and disrupt normal
physiological processes. In HHT, one of the physiological consequences is essentially excessive bleeding
and poor capillary exchange.

Lecture XVIII
C ard io va sc ular Sys tem : O rga niza tion , Fu nc tio ns, Prop er ties

Terminology
Term
Transport and Exchange
Closed versus Open
Circulation

William Harvey
Pulmonary Circulation
Systemic Circulation
A' Series
B'
In
A'

B'

Definition
Major function of the cardiovascular system, involving movement of a media
(particularly blood) through a circulatory system to allow for tissue exchange
of nutrients and metabolic waste.
Closed Circulation: Blood never leaves the network of arteries, veins and
capillaries, with oxygen and nutrients diffusing into interstitial fluid. Allows
for diffusion of nutrients at low pressure states.
Open Circulation: Blood leaves network of arteries, veins, and capillaries in
the form of hemolymph, which provides no distinction between blood and
interstial fluid. Allows for movement at higher pressure.
English physician who first described the systemic circulation and properties
of blood in a closed circulation.
Portion of the cardiovascular system carrying oxygen-depleted blood away
from the heart to the lungs, to bring oxygenated blood to the heart.
Portion of the cardiovascular system where oxygenated blood is carried to the
various organs (excluding lungs) and brings oxygen-depleted blood to the
heart.
Type of organization consisting of consecutive elements, such as A B.

Components'in'series'

InComponents'in'series'
Parallel

A'
B'
A'
B'
C'
C'
Components'in'parallel'
Artery
Components'in'parallel'

Vein
Microcirculation

Type of organization consisting of one attending to multiple events, such as A

B + C + D (simultaneously).
Blood vessels that carry blood away from the heart.
Blood vessels that carry blood towards the heart.
Small vessels in the vasculature that are embedded in organs and are the
main element in the distribution of nutrients from blood to tissue.

Arteriole
Venule
Capillary
Atrium
Ventricle
Myocardium
Atrioventricular Valves
Cardiac Output

Blood vessel in microcirculation that moves out of the artery and into the
capillaries. They are also the primary sites of vascular resistance, where the
greatest change in blood pressure and velocity of blood flow occurs.
Blood vessel in microcirculation that return blood from the capillary to the
larger blood vessels.
Smallest blood vessels and are involved in exchange of nutrients and
materials, and connect arterioles to venules.
Chamber of the heart involved in receiving blood. The right atrium receives
blood rom the superior or inferior vena cava, while the left atrium receives
blood from the pulmonary veins.
Large chamber that collect and expel blood from the atrium towards either
lungs (right ventricle) or to the body (left ventricle). The left ventricle is
typically bigger than the right ventricle.
Muscular layer of the heart involved in contraction and pumping of blood,
consisting of cardiac muscle.
Valves of the heart between the atria and ventricle, known as the mitral valve
(left) and the tricuspid (right).
Volume of blood being pumped by the heart, by a left or right ventricle within
one minute. It is the multiplication product of stroke volume and heart rate.

Q=SV HR .

Total Cross-Sectional Area

Velocity of Blood Flow

Total cross sectional area of all blood vessels of a particular type. Capillaries
(through their numbers) have the largest cross sectional area despite their
small size.
A value equal to the total volume flow divided by the cross-sectional area:

Blood Flow Velocity=

Volumetric Flow Rate
Blood Pressure
Arteriovenous
Malformation
Telangiectasia

Total Volume Flow

Crosssectional Area

Volume of fluid that passes through a given surface per unit time.
Pressure exerted by circulating blood upon the walls of blood vessels.
Abnormal connection between veins and arteries, typically from congenital
origins.
Small, dilated blood vessels near the surface of the skin or mucous
membranes.

Lecture Objectives
Know the functions of the CV system.
The cardiovascular system has the following major functions: (1) transport and exchange of nutrients, (2)
fluid balance to regulate intracellular and extracellular volume of body cells, (3) dissipation of excess heat,
(4) as a buffer system to stabilize pH. The ultimate goal of this is to maintain homeostasis, or a relatively
constant environment around cells.
Learn how the CV system is organized.
The cardiovascular system is organized in a closed circulation, in which
blood is contained within vessels (of varying size and thickness) at all
times. The components of the human cardiovascular system are: (1) the
heart, (2) blood vessels, and (3) blood. The heart is the muscular pump
that provides the driving force of the movement of blood. The blood
vessels are ultimately the plumbing of the heart, directing movement and
keeping the blood confined. Finally, the blood is the tissue involved in
carrying materials.
On a larger scale, it is organized into two circulations: (1) the pulmonary circulation (involving the right
side of the heart) and (2) the system circulation (involving the left side of the heart). The systemic
circulation is typically in series with the pulmonary circulation, while the other organ systems are supplied
with blood in parallel by the systemic circulation. It can also be organized by function. Lungs represent a
site of gas exchange. The heart is the pump. The
Superior/Inf
Superior/Inf
arteries are the distributing tubes. The capillaries
erior Vena
erior
Vena
Cava
Cava
are the large networks of very thin tubes where
Systemic
Right
Systemic
Right
Circulation
Atrium
exchange occurs at tissues. Veins are the
Circulation
Atrium
collecting tubes.
Trace the flow of blood through the CV
system.
The flow of blood through the cardiovascular
system is typically in the following manner. Blood

Aorta
Aorta

Right
Right
Ventricle
Ventricle

Left
Left
Ventricle
Ventricle

Pulmonary
Pulmonary
Arteries
Arteries

4
Left
Atrium
Left Atrium

Lungs
Lungs
Pulmonary
Pulmonary
Veins
Veins

flows into the right atrium from the superior and inferior vena cava and consequently flow into the right
ventricle. From the right ventricle, blood will then flow (via the pulmonary arteries) to the lungs, where
they will be replenished with oxygen while simultaneously releasing carbon dioxide. The newly
oxygenated blood will then flow into the left atrium via the pulmonary veins. It will then flow into the left
ventricle, and then be pumped into the aorta and thus into the systemic circulation.
Know about cardiac output and its distribution and how it can change.
Cardiac output is the volume of blood pumped by each ventricle per minute (approximately 5
liters/minute). The cardiac output from right to left ventricles is typically the same (at 5 liters/minute as it
is a closed circulatory system). If not the same, it may imply backup of flow. The
distribution of the cardiac output to the different organ systems is rather unequal,
determined
Distribu:
on'of'the' by function, size, and metabolism. Some changes in cardiac output are
rather normal, especially in states of exercise, where the metabolic demands (by
cardiac'output'to'the'
neurological excitation) require greater flow of blood.
different'organ'systems'
What$determines$
the$ parameters change at different levels of the circulation.
Learn
how CV
distribu=on$of$the$
Think
cardiac$about
output?$ how these might change in HHT.
The cardiovascular parameters can change at different
levels of the circulation. In terms of total cross-sectional
Sherwood'Fig.'10T
area,1'the capillaries (collectively) have the greatest
cross-secitonal area, because of their numbers. The
total cross-sectional area is the key determinant of the velocity of blood flow.
Typically, as the cross sectional area increases the velocity of flow decreases
(at constant blood flow rate). This is important because we can also tell that
the pressure is the lowest at the capillaries, while highest at the aorta. The
mean arterial pressure remains constant through larger arteries and then
drops at the arterioles and troughs at the venules and veins. This can change in hereditary hemorrhagic
telangiectasia (where direct connections allow paths of lower resistance) by making venous pressure
increase. People with hereditary hemorrhagic telangiectasia can have arteriovenous malformations that
spur a shunt (a low resistance shortcut) between the arteries and veins. The problem is that the veins are
not as structurally stable in comparison to the muscular arteries, and can easily hemorrhage because of
such relatively extreme pressures by such bypasses. Patients with this same disorder can develop a shunt
in the hepatic circulation, with the chronic high cardiac output that can spur cardiac failure.

Lecture XIX
B lo od Ves sel S tr uc tu re and Fu nc tion

Terminology
Term
Tunica Interna
Tunica Media
Tunica Externa
Vascular Endothelium
Vascular Smooth Muscle
Basement Membrane
Elastin
Collagen fibers
Arteries
Pressure Reservoirs
Pulsatile Blood Pressure

Definition
Most interior layer of the blood vessel, which includes endothelial cells and
connective tissue.
Medial layer of the cell, containing vascular smooth muscle cells in loose
connective tissue and elastic fibers.
Most external layer of the blood vessel, which is essentially a connective
tissue sheath, also known as an adventitia.
Thin layer of cells that line the interior surface of blood vessels, with the
individual cells called endothelial cells.
Smooth muscle found within, and composing the majority of the wall of blood
vessels, particularly the arteries and arterioles.
Sheet of fibers that underlie the epithelium, which line cavities and surfaces
such the endothelium.
Protein in elastic connective tissue to allow consistency in shape after
expansion or contraction.
Major component of connective tissue that allows maintenance in shape.
This is seen in greater amounts in venules and veins.
Blood vessels that carry blood away from the heart, exhibiting more smooth
muscle in comparison to veins.
Type of reservoir in which pressure, not volume, is the main element
regulated. This is seen in arteries, where the mean arterial pressure is kept
constant.
Quantity of pressure required creating the feeling of a pulse.

Systolic Pressure
Diastolic Pressure
Mean Arterial Blood
Pressure

Maximal blood pressure, or the pressure exhibited in contraction.

Minimal blood pressure, or the pressure exhibited in relaxation.
Average arterial pressure during a single cardiac cycle, or the average blood
pressure in an individual. Calculated as the product as the sum of the
product of cardiac output and stroke volume and central venous pressure
[ (CO SVR )+ CVP=MAP .

Veins

Blood vessels that carry blood towards the heart, exhibiting more collagen in
comparison to arteries.
Type of reservoir where volume, not pressure, is under regulation. Veins are
considered volume reservoirs, retaining the majority of blood volume.
Distribution of blood is where volume changes to different parts of the body
despite the constant pressure gradient.
Main force involved in allowing blood to flow towards the heart
unidirectionally.
Smaller vessels in vasculature involved in distribution of blood within tissues.
Blood vessel in microcirculation that moves out of the artery and into the
capillaries. They are also the primary sites of vascular resistance, where the
greatest change in blood pressure and velocity of blood flow occurs.
Blood vessel in microcirculation that return blood from the capillary to the
larger blood vessels.
Volume of fluid that passes through a given surface per unit time. It is
proportional to the pressure gradient across the vessel and is inversely

Volume Reservoirs
Distribution of Blood
Volume
Venous Valves
Microcirculation
Arterioles
Venules
Volumetric Flow Rate

related to the resistance to blood flow:

Pressure Gradient
Resistance to Flow

Poisseuilles Law

Vasodilation
Capillaries

P
R

Difference in pressure along a blood vessel.

Despite a constant pressure, the major variable involved in altering volume
and distribution of blood. It determines how much blood flow goes through a
particular blood vessel, and also determines how much flow goes into a
particular organ.
Physical law that provides a pressure drops along a blood vessel. Calculated
by:

Smooth Muscle Cell

Contractility
Primary Resistance
Vessels
Vasoconstriction

Q=

Q=

P r2
8 L

, where r is radius, P is pressure, is viscosity, and L

is length.
Ability of the smooth muscle to relax or contract independent of volume.
Vessels involved in altering blood volume despite constant pressure.
Arterioles are the primary resistance vessels.
Narrowing of blood vessels from contraction of smooth muscles, found
particularly in larger arteries and arterioles.
The widening of blood vessels from relaxation of smooth muscles.
Smallest blood vessels and are involved in exchange of nutrients and
materials, and connect arterioles to venules.

Lecture Objectives
Learn about the anatomy of blood vessels.
The anatomy of blood vessels is distinctly different among the arteries and veins. Remember that their
structure is reflective upon function, but as blood vessels (excluding
the capillaries), they have similar parts. The blood vessel contains
three parts: the tunica interna, the tunica media, and the tunica
externa. The tunica interna is the innermost layer of the blood
vessel, containing the endothelium and connective tissue. All blood
vessels are lined with the endothelial cells, which function in clot
prevention, cell signaling, and capillary exchange. The tunica media
is the smooth muscle layer, containing the smooth muscle in loose
connective tissue with elastin. Elastin is important in allow the recoil
from the larger volume of blood flow. The tunica externa (also
known as the adventitia) represents a connective tissue sheath that
encapsulated the blood vessel.
Though arteries and veins are classified under blood vessels, they
have observable differences. Arteries have a larger amount of smooth muscle and elastin, while veins
tend to have greater amounts of collagen fibers. Thus, compensate in order to establish unidirectional

flow, the veins are accommodated with valves made from endothelial tissue.
is simply because they are one cell thick and are the main
exchange points between the blood and tissue.

The exclusion of capillaries

See how the anatomy of different types of blood vessels

reflects function.
The anatomy is reflective upon function. Arteries typically move
blood away from the heart, while veins move blood towards the
heart. Their function can be represented structurally in the fact
that arteries have larger amounts of elastin and vascular
smooth muscle lining this type of blood vessel. This allows the
artery to propagate pressure waves from the heart (allowing
transient increases in blood), and allow it to recoil back from
expansion. Thus, the arteries are treated as the pressure
reservoirs of the circulatory system, keeping the mean arterial pressure constant and allow even pressure
distribution to the system.
Meanwhile, veins display a different structure for their function. They are involved in
carrying tissues back to the heart. They have thinner walls compared to arteries and
less elastic tissue and smooth muscle, and are typically adapted for lower pressures.
To facilitate venous return to the heart in a unidirectional fashion, they have a series of
valves. Thus, they are typically considered to be volume reservoirs, as they are the
major storage of blood volume (containing approximately 64% of volume distributed).
Know the functions of endothelial cells and smooth muscle cells.
The endothelial cells of the body are involved in (1) provision of a smooth interface, (2)
secretion of cellular signals, and (3) in the capillaries, provide a penetrable barrier for
exchange.
Smooth muscle cells in the vasculature can contract or relax depending on what kinds
of signals they receive. Arterioles are the major resistance vessels. A change in
arterioles will cause a change in flow. The largest drop in pressure occurs at the
arterioles, and arterioles control the flow of blood amid the constant pressure. They typically act by
vasoconstriction and vasodilation. Vasoconstriction occurs by increased contraction of smooth muscle,
where there is increases in resistance and eventual decrease in flow. Vasodilation occurs when there I a
decreased contraction of smooth muscle, where there is a decrease in resistance and an increase in flow.
Resistance is what determines how much flow goes into each organ.
Local and extrinsic factors can affect the activity of the vascular smooth muscle. It can be affected by (1)
autonomic nervous system activity, such as in state of physical activity, (2) local factors, or metabolites,
released by the nearby tissue, giving a metabolic control of blood flow, (3) local changes in blood flow,
showing myogenic control, and (4) signals released by the vascular endothelium, such as nitric oxide and
endothelin.
Know the different parameters that affect blood flow through a blood vessel.
There are several general parameters that can affect blood flow through a blood pressure, as Poiseuilles
Law can describe. We do know that pressure and resistance are the two major variables that can affect
flow, but there are others. The radius is inversely proportional to resistance, such that an increase in the
radius can cause a decrease in resistance. However resistance is directly proportional to the viscosity and
length, so that increasing either viscosity or length can cause an increase in resistance.
However, we should also examine the role of blood pressure. It is
not dependent on the absolute values of pressure along the blood
vessel, but more of the difference between the pressures along the
blood vessel, which is known as a pressure gradient. In physiological
states, however, we should remember that there is going to be
change in blood vessel structure as the absolute pressures increase.
Think about how the above relate to the case study. What changes occur with HHT?
In hereditary, hemorrhagic telangiectasia, we should remember that the arteriovenous malformations
allow a low resistance bypass into the vein. Higher pressures in the draining veins at focal vascular lesions
in HHT can lead to consequential structural changes in the veins. However, veins are particularly thinner
relative to arteries, and the compensatory measures are inadequate. If the pressure is too high and the
structural compensations are insufficient, it can lead to strokes, aneurysm, and hemorrhage due to such
structural insufficiency. The laminar flow is disrupted in the arteriovenous malformation and spur turbulent

flow. Such turbulent flow can cause structural changes and greater proliferation to accommodate such
changes in flow.

Lecture XX
Transp or t a nd E xch ang e in the Micr oc ir cu la tio n

Terminology
Term
Microcirculation
Arterioles
Metarterioles
Precapillary sphincters
Venules
Capillaries

Capillary Recruitment
Continuous Capillaries

Definition
Smaller vessels in vasculature involved in distribution of blood within tissues.
Blood vessel in microcirculation that moves out of the artery and into the
capillaries. They are also the primary sites of vascular resistance, where the
greatest change in blood pressure and velocity of blood flow occurs.
Short vessel that links arterioles to capillaries, which contain smooth muscle
cells a short distance apart that forms a precapillary sphincter that surrounds
entrance to capillary bed.
Band of smooth muscle that adjusts flow into capillary.
Blood vessel in microcirculation that return blood from the capillary to the
larger blood vessels.
Smallest blood vessels and are involved in exchange of nutrients and
materials, and connect arterioles to venules. Capillaries are suited as a site
of exchange because of the minimal diffusion distance, maximal surface area,
and the maximal time for exchange.
Increase in the number of perfused capillaries in response to stimuli.
Type of capillary that has an uninterrupted lining in the endothelial cells, to
allow water and ions to diffuse with two types of tight junctions: (1) one with
many transport vesicles such as those in skeletal muscle, and (2) those with
few vesicles, such as in the central nervous system.

Fenestrated Capillaries

Type of capillary with pores in the endothelial cells that allow small molecules
and small proteins to diffuse, found in kidneys and exocrine glands.

Sinusoidal Capillaries

Type of capillary with larger openings in the endothelium due to a

discontinuous basal lamina, which allow cells and larger proteins to enter.
These are found in bone marrow, lymph nodes, liver, spleen and adrenal
gland.

Diffusion

Movement of particles from areas of higher concentration to areas of low

concentration.
Mathematical relationship in diffusion:

Ficks Law

Net rate of diffusion ( Q ) =

Concentration Gradient
Lipid-Soluble Substances
Small, Water-Soluble
Substances
Exchangeable Proteins
Plasma Proteins
Endocytosis
Exocytosis
Secondary Polycythemia
Interstitial Fluid

CP A
MW X

, where C is concentration, P

is permeability of membrane to substance, A is surface area of membrane,

MW is the molecular weight of substance, and X is distance.
Gradual difference in the concentration of solutes in a solution between two
regions. Rate of diffusion depends on the concentration gradient.
Nonpolar substances that can dissolve in fats thus can pass through the
phospholipid bilayer.
Polar or charged solutes that need to be dissolved in water thus will diffuse
across certain pores or gaps in cells.
Proteins that are shuttled across the membrane by a general vesicular
mechanism of transport, such as endocytosis and exocytosis.
Proteins that are generally too large to pass through the water-filled pores
thus remain in the plasma.
Cells absorb molecules through engulfment.
Cells direct contents of solutes out of the cell membrane via secretory
vesicles.
Type of increase in erythrocytes either by natural or artificial causes, with a
known underlying cause.
Fluid that bathes and surround cells of tissue, and is the major component of

Plasma Fluid
Intracellular Fluid
Bulk Flow
Ultrafiltration
Reabsorption
Lymphatics
Net Fluid Exchange
Pressure

extracellular fluid.
Liquid component of blood where blood cells in whole blood are suspended.
Fluid found inside cells, which is also known as cytosol or cytoplasm.
Movement of a fluid driven by pressure.
Type of membrane filtration involving hydrostatic pressure pushing against a
semipermeable membrane. In this case, there is a push for fluid out of the
capillary. Typically, ultrafiltration is larger than reabsorption by 3 L.
Type of membran filtration in which there is a uptake of fluid into the capillary.
Organ system involved in filtration of lymph and reuptake of fluid that is not
reabsorbed into the capillary. Consist of blind-ended capillaries that overlap
the capillary system to transport fluid back into the venous system.
Contributor of bulk flow which is the difference in the outward and inward
pressures, such is also influenced by derivatives of the outward and inward
pressure, such as capillary pressure (PC), interstitial fluid pressure (PIF),
plasma colloid osmotic pressure (P), and the interstial fluid colloid osmotic
pressure (IF). Thus:

Capillary Pressure
Interstitial Fluid Exchange
Pressure
Plasma Colloid Osmotic
Pressure
Interstitial Fluid Colloid
Osmotic Pressure
Starlings Law

P=P outwardPinward =( PC + IF )(P IF + P)

Outward force for fluid exchange.

Inward force for fluid exchange.
Inward Force for fluid exchange.
Outward force for fluid exchange.
Mathematical illustration of hydrostatic and oncotic forces to determine
amount of fluid movement from blood to tissue. Calculated as:

Qf =L p SP=L p S [ ( PC + IF ) (P IF + P )]

. Exchange pressure

determines direction of movement, while both exchange pressure, and

permeability will determine magnitude of the movement.

Lecture Objectives
Know the organization, anatomy, and function of the microcirculation.
We need to remember that microcirculation is the site of exchange of solutes and
fluid. The microcirculation consists of three general anatomical parts: the
arteriole, the capillaries, and the venule. On the side of the arterioles, there are
smooth muscle cells in areas such as the metarteriole and the precapillary
sphincter that allow regulation of flow through the capillaries. It should also be
remembered that the capillary is one-cell thick to allow a site of exchange.
Learn how the exchange of solutes occurs in capillaries.
The exchange of solutes between the plasma and tissue cells occurs via a
mechanism known as diffusion in the capillaries. Diffusion is the movement of
solutes from areas of high concentration to areas of low concentration. One can
calculate the net rate of diffusion Q by Ficks Law:

Net rate of diffusion ( Q ) =

CP A
MW X

Tissue'metabolic'ac8vity'

, where C is

O2,''CO2','other'metabolites'

Effect'of'capillary'
recruitment'and'
arteriolar'dila8on'

concentration gradient, P is permeability of membrane to substance,

Relaxa8on'of'
Arteriolar'vasodila8on'
precapillary'sphincters'
A is surface area of membrane, MW is the molecular weight of
substance, and X is distance. Capillries are well suited as a site of
Capillary'blood'flow'
exchange because of the minimal diffusion distances (low X),
Number'of'open''
capillaries'
Delivery'of'O ,'more'
maximal surface area (large A), and a low flow rate (giving it a lot of
rapid'removal'of'CO '
and'other'metabolites'
time for the necessary exchange to occur, because flow is inversely
Diffusion'distance''
Concentra8on'gradient''
Capillary'surface'
proportional to cross-sectional area). However, at any particular
from'cell'to'open''
between'
area'available'for'
capillary'
blood'and'8ssue'cells'
exchange'
moment, not all the capillaries are open. In skeletal muscle, only
10% of the capillaries are open. In more physical states, such as in
Sherwood'Fig.'10X20'
Exchange'between'blood'
and'8ssue'
exercise, metabolites diffuse in the precapillary sphincters and allow
blood to flow into the capillaries due to dilation (relaxing) of the
sphincters and arterioles. The major strategy of this is to maintain the concentration gradient. Capillary
recruitment is also self-regulatory, because decreases in physical activity will cause a decrease in flow and
removal of the vasodilating metabolites and cause the precapillary sphincter and arterioles to contract.
2

In the capillary, the exchange of solutes is known as transcapillary exchange, in which the solutes will
move based on the intrinsic diffusion characteristics. Lipid-soluble substances, such as O 2 and CO2, will
diffuse across the membrane because they are chemically nonpolar and are able to dissolve in lipids.

Other substances, particularly those that are water-soluble and small, need to be dissolved in water first,
but can diffuse across gaps in cells. Remember, the rate of diffusion is dependent on the concentration
gradient, size of the solute, and the polarity of the solute. With this, there are also three different types of
capillaries, the (1) continuous (which is the most common) and found in skeletal muscle, skin, the (2)
fenetrated, which is found in kidneys and exocrine glands for filtration purposes, and (3) sinusoidal, found
in the liver, spleen, where there is necessary movement of cells and larger proteins. The capillary pore
sizes vary in different tissues. In general, permeability to small solutes is greater than permeability to
large molecules. However, the capillary permeability can be altered under certain conditions, such as
histamine (an inflammatory response chemical released by mast cells that increases the size of the
capillary pores). For larger molecules, such as proteins, diffusion is not the optimal mechanism, so
vesicular mechanisms such as endocytosis and exocytosis are utilized to shuttle proteins to the interior or
the exterior of the cell. So, we can summarize the factors of transcapillary exchange of small solutes in
the following diagram:
Factors affecting
transcapillary exchange of
small solutes

Concentration gradients:
Determines direction of
movement and magnitude of
movement of solutes

Volumetric Flow Rate:

Maintains fresh diffusion
gradients

Velocity of blood flow:

Determines time available
for exchange by diffusion.

Capillary surface area: The

greater the number of
capillaries open, the greater
the surface area for
diffusion.

Permeability of Capillaries to
Solutes: The size of the
water-filled pores between
endothelial cells is
important.

d 'exchange'pressure''
Know how exchange of fluid occurs in capillaries and learn about StarlingsDeterminants'of'net'flu
Law.
The movement of solutes occurs through the interstitial fluid, which serves as a
Plasma'colloid'
Capillary'
osmo8c'pressure'
pressure'
i (PC)'
passive intermediary. Interstitial fluid is the majority of extracellular fluid
(P)'
(approximately 80%) and serves a homeostatic role of the cell. Intracellular fluid
constitutes the majority of fluid, but plasma fluid and interstitial fluid play a role in
Inters88al'fluid'
Inters88al'fluid'
pressure'(P )'
colloid'osmo8c'
providing the optimal environment for cells. The exchange of fluid between
pressure'(IF)'
capillaries and interstitial fluid typically occurs via bulk flow.
Inters88al'fluid'colloid'osmo8c'pressure'is'an'
Typical'values..'
In bulk flow, there are two movements: (1) ultrafiltration, in
Ini8al'lympha8c'
outward'force'for'fluid'exchange'
vessel'
which the fluid moves out of the capillaries, and (2)
reabsorption, in which the capillaries take up the fluid.
Inters88al'
Ultrafiltration is typically greater than reabsorption by
flud'
11'mm'Hg'
9'mm'Hg'
approximately 3 liters a day, but the fluid is returned to the
(ultrafiltra8on)'
(reabsorp8on)'
plasma by the lymphatic system, which transports the
excess fluid (known as lymph) back into the venous system.
Bulk flow is influenced by net fluid exchange pressure,
which is an outward and inward pressure difference. Four
forces determine them: capillary pressure (outward),
From'
Blood'
To'
interstitial fluid pressure (inward), plasma colloid osmotic
arteriole'
capillary'
venule'
pressure (inward), and interstitial fluid colloid osmotic
pressure (outward). Thus, we can calculate the net fluid exchange pressure:
IF

P=P outwardPinward =( PC + IF )( P IF + P)

Sherwood'Fig.'10X22'

. From there, we can determine the fluid

movement between blood and tissue, which is known as Starlings Law/Equation, which is:

Qf =L p SP=L p S [ ( PC + IF ) (P IF + P )]

. From there, we can draw two conclusions: (1) the

pressure difference determines the direction of fluid movement, and (2) either fluid permeability
coefficient, capillary surface area, or pressure difference will determine the magnitude of the fluid
movement.
Discuss how the above relate to the case study.
In hereditary hemorrhagic telangiectasia, the transfer of small solutes can be locally affected at the site of
the vascular lesions. Thus, the decrease in number of capillaries can spur less exchange occurring at the
local areas. If there is a pulmonary arteriovenous malformation in a patient, individuals can develop
secondary polycythemia, which is essentially an elevated hematocrit. This response is mainly to
compensate for the low oxygen concentration in the blood. Thus, the adrenal glands secrete
erythropoietin to increase carrying of oxygen to the tissues in response to the presence of the pulmonary
arteriovenous malformation. In terms of pressure, one can relate the low-resistance bypass as not
beneficial because they do not retain the properties that allow for optimal exchange (which is the complete
opposite of a capillary). Thus there is little diffusion across the membrane in the between the capillary and
lung or between the capillary and tissues. This is why patients with HHT often will present with fatigue and
secondary polycythemia.

Lecture XXI

10

Vas cu lar D evelop ment

Terminology
Term
Cell Differentiation
Cell Proliferation
Cell Movement
Cell Interaction
Inductive Interaction
Gene Expression
Vasculogenesis
Angiogenesis
Angioblasts
Mesoderm
Yolk Sac

Blood Islands
Primary Capillary Plexus
Embryonic versus ExtraEmbryonic Vasculature
Vascular Endothelial Cell
Growth Factor (VEGF)
Receptor Tyrosine Kinase
Transphosphorylation
Angiogenesis
Sprouting Angiogenesis
Non-Sprouting
(Intussusceptive)
Angiogenesis
Activation Phase
Resolution Phase
Basement Membrane
Mesenchymal Cells
Tip Cells
Platelet-Derived Growth
Factor (PDGF)
Mural Cells

Definition
Process by which a less specialized cell becomes more specialized in a
multicellular organism.
The growth or production of cell by multiplication of parts
Movement of the cell in response to
Direct interactions between cells that play a role in development and function
of multicellular organisms.
Interaction between two groups of cells in which a signal passed from one
group of cells causes the other group of cells to change their developmental
state (or fate).
Process by which information from a gene is used to synthesize a protein or
gene product.
Differentiation of angioblasts into endothelial cells and their assembly into a
primary vascular plexus. Formation of the vascular network in some organs
occurs mainly by vasculogenesis.
The process by which new vessels form by sprouting or splitting of preexisting vessels. In other organ systems, angiogenesis may be more
prominent.
Differentiated cell from the mesoderm formed from hemangioblasts.
Primary germ cell layer that yields the cardiovascular system. In response to
BMP, they form hemangioblast, which eventually forms parts of the
vasculature.
Membranous sac attached to the embryo, providing nourishment to the
embryo. Around the 4th week of embryonic development, the functioning
vasculature is present in the yolk sac, and angioblasts develop in the blood
islands.
Structures in the developing embryo that can yield different parts of the
circulatory system, derived from plexuses formed from angioblasts.
Initial site of vascular remodeling. It is formed by vasculogenesis.
Embryonic: Involving angioblasts to form tubes that yield mature vessels.
Extra-Embryonic: Involving angioblasts developing blood islands, which form
a primary plexus to yield a mature vessel.
Signaling protein produced to stimulate vasculogenesis and angiogenesis.
High-affinity cell surface receptors for many polypeptide growth factors,
cytokines, and hormones. Play a critical role in binding of VEGF for
angiogenesis and vasculogenesis.
Movement of one phosphate group of one molecule to another molecule.
VEGF causes receptor dimerization and transphophorylation of tyrosine
residues.
Process by which new vessels form by sprouting or splitting of preexisting
vessels.
Form of angiogenesis in which tip cells (designated endothelial cells) extend
into the extracellular matrix creating vessels.
Form of angiogenesis in which the capillary wall extends into lumen to split
the vessel into two.
Phase in which vascular permeability and degradation of basement
membrane allowing endothelial cells to migrate into the matrix to proliferate.
Endothelial cells discontinue growth and migration and allow reformation of
basement membrane.
Sheet of fibers that underlie the endothelium of the vasculature.
Multipotent stem cells that can differentiate into different cells. In the
presence of activated TGF-, mesenchymal cells differentiate into mural cells,
which consequently differentiate into vascular smooth muscle or pericytes.
Designated endothelial cells that are the point men of the activation phase.
Specific inductive signaling occurs to regulation formation and migration of
tip cells, particularly VEGF-A and Notch signaling.
One of many growth factos involved in regulation of cell growth and division.
Vascular smooth mucle cells or pericytes that are involved in the formation of
normal vasculature, which respond to VEGF.

11

Transforming Growth
Factor (TGF-)
Postnatal Angiogenesis

Protein that controls proliferation, differentiation in of endothelial cells.

Important in vessel maturation and balances proliferation and differentiation.
Angiogenesis that occurs after the birth and later development of the human,
which can occur in states of hypoxia and trauma.

Lecture Objectives
Know some general principles of development.
The general principles of development typically involve starting
simple and simply refining. Vascular development involves a series
of maturation and refinement, and recruitment of other cells for the
associations, leading to stabilization and maturation. The
remodeling and maturation then allows formation of vasculature,
with tissue-specific accommodations. This process occurs in the
embryo. The angioblasts, the precursor cells, become defined to
move from vasculature without forming intermediate primary plexus.
There are two major phases of vascular development: (1)
vasculogenesis and (2) angiogenesis. Vasculogenesis is the
formation of blood cells from undifferentiated cells to tube.
Angiogenesis involves formation of vessels from pre-existing vessels,
which can involve splitting of pre-existing vessels.

Nature$438,#937-45#(2005).

Vascular development involves the differentiation, proliferation,

and migration of cells that will form the blood vessel tubes
(endothelial cells). Within this process, there is a formation of a
primitive network of tubes of endothelial cells known as the
primary capillary plexus. Recruitment and differentiation of
mural cells will yield smooth muscle cells or pericytes. From
this, sprouting, branching, intussusception and remodeling
occurs to form a more mature network, with eventual
further maturation of blood vessels to reflect specific
functions of vessel types.
These processes are mediated by cellular signaling.
Multicellular animals have proteins (that are typically
conserved) that are involved in the mediation of cellular
interactions and signal transduction. The inductive signals
are important to spur orderly differences from initially
identical cells. Signals are typically involved in gene
expression, meaning that the genes are in control of the developmental process, sometimes repeatedly.
Learn about the process of vasculogenesis during embryonic development.
Vasculogenesis is considered the first phase in vascular development. Endothelial cells are derived from
precursor cells, known as angioblasts, that originate from the mesoderm. Remember that the mesoderm
gives rise to the cardiovascular system. By approximately the fourth week of embryonic development,
there is a functioning vasculature, and nutrients are from the placenta as well as the yolk sac. Angioblasts
typicaly develop from the blood islands of the yolk sac. From this, a primary capillary plexus in the yolk
sac is formed by vasculogenesis and then goes through remodeling. The larger vessel is giving rise to a
smaller vessel. On top of this, the angioblasts arise in the embryo proper as well as migrate out of the
mesoderm in somites and form vessels in various parts of the body, including part of the aorta. They will
eventually spur formation of blood vessels. However, there is such a thing as too much of a good thing in
this case. Obviously, having not enough angioblasts can spur poor formation of the cardiovascular system,
but having too much can compromise the embryo due to nutrient depletion and excessive remodeling.
Thus, the embryo must regulate the formation of angioblasts.
One factor that is important in vasculogenesis is known as vascular endothelial cell growth factor (VEGF).
They promote angioblast formation and proliferation and allow formation of blood vessels by inductive
interaction.
Inductive interaction is a stepwise interaction between two groups of cells in which a signal passed from
one group of cells causes the other group of cells to change their evelopmental state (or fate). VEGF binds
to a receptor tyrosine kinase, in which the binding of VEGF to the extracellular domain causes protein
changes to spur a signaling cascade and desired effect. Though there are different types of VEGFs and
associated receptors, there are different effects present that are dependent on the receptor (not on the
factor). The receptor dictates the effect of the binding, NOT the factor. VEGF can promote vasculogenesis
and angiogenesis, but also lymphangiogenesis (formation of lymphatic vessels). VEGF causes the receptor
dimerization and transphosphorylation of tyrosine residues, similar to growth factors such as EGF, and

12

There'are'different'types'of'VEGFs'and'VEGFRs'

Signaling Pathways Activated by VEGF

Vasculogenesis'
Angiogenesis'

Lymphangiogenesis'

causes attraction of proteins to the signaling complex as well as

recruitment of different enzymes for the signaling pathway. Different
effects can occur depending on the signaling pathway by VEGF. VEGF
can ensure (1) cell survival, (2) gene expression and cellular
proliferation, (3) cellular proliferation and vasopermability, which
ultimately leads to (4) vasculogenesis and angiogenesis.

SIGMA-ALDRICH

Learn about the process of angiogenesis during embryonic

development and postnatal angiogenesis.
Angiogenesis, as stated before, is the growth of blood
vessels from preexisting vessels. The second phase of
vascular development has two types: (1) non-sprouting
Cell'survival'
(or intussusceptive) angiogenesis and (2) sprouting
Gene'expression'
Cell'proliferaPon'
Cell'proliferaPon'
Vasopermeability'
Focus'on'the'
angiogenesis. They typically can either sprout from the
physiological'
primary plexus or be non-sprouting. Intussusceptive
effects'here'
Vasculogenesis'
Angiogenesis'
angiogenesis typically presents with a formation of a
blood vessel pillar that splits off into two different vessels. The holes will eventually connect
with each other to produce a much finer network. In some body regions (such as the limbs),
the blood vessels develop during embryogenesis mainly by angiogenesis not by
vasculogenesis.
Sprouting angiogenesis involves two phases: (1) Activation
and (2) Resolution. Activation involves the preexisting tube
to sprout out and form tip cells. In the Activation Phase,
cells loosen connections and degrade the extracellular
matrix to allow for proliferation and migration from tube. In
this phase, vascular permeability increases, degradation of
the basement membrane is permitted to allow endothelial
cell migration and proliferation. Tip cells are the endothelial
point men that move in the Activation Phase. The specific
inductive signaling occurs to regulate formation and
migration of tip cells, involving VEGF-A signaling and the
Notch signaling pathway. VEGF-A is the signal that
designates the endothelial cell to be a tip cell and sprout.
The Notch Signaling Pathway is simply a preventive tool
involving lateral inhibition of neighboring endothelial cells
to prevent unnecessary sprouting in proximity of the
designated tip cell. The Resolution Phase involves
reformation of the extracellular matrix as well as recruitment and differentiation of mural cells to become
endothelial tubes. Endothelial cells halt migration and proliferation, the cell-cell junctions are tightened,
and reformation of the basement membrane occurs, allowing mesenchymal cells to be recruited to
differentiate into mural cells. At this time, the pericytes (which are sparsely covered) involves the
recruitment of mesenchymal cells to migrate and associate with the newly developed blood vessels.
Recruitment and differentiation of mural cells to the endothelial tubes involves PDGF- secretion from the
tip cells to recruit mesenchymal cells to migrate and contact endothelial cells. The cell-cell contact results
in activation of latent transforming growth factor (TGF-) in the extracellular matrix. The activated TGF-
causes mesenchymal cells ot differentiate into the mural cells (either vascular smooth muscle cells or
pericytes). TGF- signaling is needed for formation of vascular smooth muscle cells, and also occurs in
endothelial cells (in a paracrine fashion) to control balance between proliferation/migration and
differentiation. Other signaling molecules are also involved in the development of new blood vessels, and
their numbers can be altered depending on the body organ involved.
Postnatally, angiogenesis can also occur, such as in states of wounding or
hypoxia. In states of wounding or vascular trauma, the factors are present
and can allow proliferation of these factors and spur a much finer network.
In states of hypoxia, VEGF is also present. Tissue cells produce a factor
known as hypoxia inducible factor that is sensitive to O2 levels in the blood.
Normally, HIF is inhibited due to high oxygen levels, but can be turned on in
low oxygen states and allow capillary sprouting through increased secretion
of VEGF.
This leads to this case study. Local hypoxia can affect the progression of arteriovenous malformation. The
hypoxia is not from the decreased concentrations of oxygen in the blood, but the poor diffusion of oxygen
into the tissues from a low-resistance bypass. Thus, there is increased production of VEGF and
proliferation of vascular endothelial cells. However, they promote the formation of vessels called torturous
vessels that worsen the arteriovenous malformation due to contortion and capillary bed destruction.

13

Look at a few selected signaling systems (VEGF, PDGF, TGF-) that are important for
vasculogenesis and angiogenesis.
Growth Factor
Acrony
Role in Vasculogenesis and Angiogenesis
m
Vascular
VEGF
Important in vasculogenesis. It binds to receptor tyrosine kinase,
Endothelial Cell
causing vasculogenesis, angiogenesis, or lymphangiogenesis depending
Growth Factor
on te receptor. Important in (1) cell survival, (2) cell proliferation and
vasopermeability, (3) gene expression, and ultimately (4)
vasculogenesis and angiogenesis.
Platelet-Derived
PDGF
Secreted by the tip cells, allow recruitment of mesenchymal cells to
Growth Factor
migrate and contact endothelial cells. Like VEGF, it acts through
receptor tyrosine kinase.
Transforming
Important in vessel maturation, and allow mesenchymal cells to
TGF-
Growth Factor, Type
differentiated into mural cells.

Hypoxia-Inducible
HIF
Allow postnatal angiogenesis in response to hypoxic states.
Factor

Lecture XXII
T GF- L igan d Sup er fa mily: Pro perties , Cell B io lo g y, Physiolog ic al Eff ects

Terminology
Term
HHT Types

Activin Receptor-Like
Kinase 1 (ALK-1)
Endoglin
TGF- Superfamily
Transforming Growth
Factors
Transformed Cells
Soft Agar Colony Growth
Assay
Anchorage-Independent
Growth
Autocrine Signaling
Paracrine Signaling
Endocrine Signaling
Apoptosis
Proteinase Inhibitors
Extracellular Matrix

Metalloproteinases

Definition
There are five major types of HHT depending on the problem area:
HHT Type I: Involves a mutation in the endoglin (ENG) gene, which is
a type III TGF receptor family co-receptor.
HHT Type II: Involves a mutation in the activin receptor-like kinase
(ALK-1) gene, which is a Type I TGF superfamily receptor.
HHT Type III: Mutation on a locus on chromosome V.
HHT Type IV: Mutation on a locus of chromosome VII.
Juvenile Polyposis/HHT Syndrome: Mutations in the gene for SMAD4,
a transcription factor.
Receptor in the TGF Signaling Pathway that allows increases in the
endothelial cell proliferation and migration.
Glycoprotein on surface of cells that participates in the TGF signaling
pathway.
Group of structurally related proteins involved in the TGF signaling pathway.
They are involved in embryogenesis, cell differentiation, cell cycle arrest, and
apoptosis.
Polypeptide growth factors that are involved in cellular proliferation and
differentiation, but have structural and functional differences.
Cells that have been altered to a cancer-like state due to the direct uptake,
incorporation, and expression of exogenous genetic material.
Type of assay in which cell colonies are grown independent of attachment to a
surface an essentially suspended in agar.
A type of growth in which cells are not suspended in a solid environment (to
allow determination of growth in certain factors by not being attached).
Type of signaling in which cell secretes a messenger
that binds to receptors on the same cell
Type of signaling in which cell secretes a messenger
that binds to receptors on a neighboring cell.
Type of signaling in which cell secretes a messenger
into the bloodstream which binds to receptors on a
distant cell.
Process of programmed cell death by certain biochemical events.
Type of inhibitor involved in the repression of extracellular matrix degradation.
Part of tissue that provides structural support to cells, consisting of the
interstitial matrix and the basement membrane. It provides support,
segregation, and regulation of intercellular communication, maintaining
stability amid the cells dynamic behavior.
Group of proteinases that are classified by the most prominent functional
group on their active site. They are proteolytic enzymes involving a metal in

14

2D Culture
3D Culture
Activins
BMPs
Agonist
Antagonist
Dorsal-Ventral Patterning
Left-Right Asymmetry
N-terminal Signal Peptide

Latency Associated
Peptide
Small Latent Complex
Large Latent Complex
TGF- Activation

their catalytic enzyme.

Culturing cells in flat plastic petri dishes (where the cells adhere to the
surface), which allows for a 2-dimensional environment for the cells.
Culturing cells in a suspension to provide a more physiologically relevant
enevironment for the cells, without attachment.
Dimer that is involved in cellular proliferation, differentiation, apoptosis,
metabolism, homeostasis, immune response, wound repair, and endocrine
function.
Type of growth factors that are interact with receptors on the cell surfafe that
are involved in growth and differentiation.
Chemical that binds to receptor of a cell that triggers a response by the cell,
typically mimicking a naturally occurring susbstance.
Type of chemical that blocks a biological response when binding to a receptor,
dampening cellular response.
Type of patterning that involves compartmentalization the embryo to account
for certain features at the dorsal-ventral locations.
No symmetry at the left and right side of the body.
The n-terminus is the first part of the protein that emerges from the ribosome
in translation. It has signal peptide sequences that spurs delivery to the
proper organelle. The peptide is removed at the destination by a signal
peptidase.
Protein derived rom N-terminal region of the TGF that interacts with the TGF
homodimer to for a complex.
Complex consisting of the N-terminal region of the TGF gene product and the
TGF homodimer.
Larger complex secreted in the extracellular matrix that consists of latent
TGF- binding to the Small Latent Complex.
Activation of latent TGF to an active state by signaling pathways or various
other factors.

Lecture Objectives
Learn about the molecular genetics of HHT.
There are several types of HHT, which can be determined via laboratory
testing involving DNA markers. The mutations in the Endoglin and ALK1 are
the most common cause of hereditary hemorrhagic telangiectasia. These
mutations can cause alterations in splicing at the donor and receptor sites
and spur a loss of function due to their scattered distribution of the key sites.
Most mutations are a loss of function, possibly haploinsufficiency from a
having at least one bad-coding copy in the genotype.
Type
I
II
III
IV
Juvenile Polyposis/HHT Syndrome

Mutation
Endoglin (ENG) Gene
Activin Receptor-Like Kinase 1 (ALK-1) Gene
Candidate Locus on Chromosome 5
Candidate Locus on Chromosome 7
Mutations in gene for SMAD4 (transcription factor)

Learn about the discovery of TGF-.

The genes that are mutated in HHT are part of the TGF- signaling pathway, in which binding to the
receptor causes a signal transduction that can alter gene expression. And remember that latent TGF
needs to be activated to spur the vascular development. This signaling pathway is one of many that can
influence gene expression.
TGF (known as Transforming Growth Factor-) was discovered about 30 years ago around the late 1970s.
It was discovered when researchers began testing the hypothesis that some tumor viruses could transform
cells into a cancer-like state by inducing cells to secrete molecules that acted on the same cells, causing
transformation via autocrine control. These factors were partially purified which could cause normal
fibroblasts to form progressively growing colonies in soft agar with reversible effects. After isolation and
purification by column chromatography and various other testing, they found two proteins that are
involved: TGF- and TGF-. TGF- is structurally related to epidermal growth factor and was the factor
involved in the growth. However, TGF- was the protein that had the growth-inhibitory effect, among
several other effects. When grown in a 2-dimensional culture (meaning that the cells attached to the flat
petri dish), there is actually an inhibition of proliferation and migration of cells. However, in the threedimensional soft agar culture, there is the formation of tube-like structures.

15

Learn about the diverse functions of the TGF-

superfamily.
The TGF- superfamily is involved in many functions:
1. Cell growth (inhibition)
2. Cell differentiation (promotion)
3. Apoptosis (induction)
4. Embryonic Development
5. Wound Healing
6. Vascular Remodeling
7. Roles in immunity, fibrosis, cancer, heart disease,
diabetes, and Marfan Syndrome
Scientists determining other functions eventually
converged upon the finding of TGF-. It contributes to
vascular development in the following ways:
1. Regulation in the proliferation, differentiation, and migration of endothelial cells and mural cells.
2. Vascular remodeling and vessel maturation
3. Stimulation in the production of extracellular matrix proteins and proteinase inhibitors (repressing
matrix degradation)
Know the general properties of TGF- superfamily ligands.
This family of proteins is often context dependent, in which the response is dependent on: (1) ligand and
receptors, (2) other signals, (3) cell type, (4) signal intensity, (5)
signal duration. However, experimentally with endothelial cells,
there is a negative feedback mechanism associated with the
context-dependent response. Low levels of TGF are associated with
an enhancement of proliferation and migration of cells, while high
levels of the same protein can inhibit the effect. One way to
regulate the levels is through binding affinity of the receptor.
The TGF-s are often associated with embryogenesis, cell differentiation, cell cycle arrest, and apoptosis.
The properties within the TGF- superfamily are similar. They are a secreted protein containing an Nterminal signal peptide (a zip code that ensures that the protein goes to the specific organelle). The Cterminal region consists of approximately 112 amino acids that become the mature TGF that is the active
ligand (as a dimer). The middle portion of the TGF- encodes the latency-associated peptide. All three of
the forms of TGF- are initially released from the cell in a latent/inactive form, and need to be activated to
achieve an effect. There are several advantages of having the latent form:
1. Inactive complex provides an opportunity for intricate regulation. Activation pathways may be
cell or tissue specific.
2. Differences in the LAPs provide different TGF complexes that are selective to specific stimuli.
3. Signaling can be locally activated by proteolysis or by mechanical strain.
Know about the process of TGF activation from a latent state.
TGF- activation involves the
latency-associated protein
combining with the active TGF- (in
various catalysts) to form the small latent complex. This complex can make further associations, by
attaching the small latent complex with latent TGF- binding protein via a disulfide bond, forming the Large
Latent Complex. This Large Latent Complex can then covalently link with the extracellular matrix. The
mature ligand dimerizes to form the active molecule. From there, there are several mechanisms to fully
activate TGF-:
1. Degradation of ECM proteins (fibrillin or LTBP) by proteases.
2. Cleavage, dissociation, or conformational change in LAP.
3. Integrin-dependent activation (by activating metalloproteases or
by a mechanical strain-dependent form).
Learn about the role of antagonists of TGF- superfamily ligands.
The antagonists of the TGF- signaling can function to establish complex
spatial patterns of functional activity because there is regulation.
Antagonists are important to establish the dorsal-ventral patterning, such
as in Noggin. Antagonists are also important for establishing left-right
asymmetry. The inhibitors can be expressed in interesting patterns. The protein involved is Lefty, a TGF-
superfamily member that inhibits Nodal signaling (another TGF- family member), helping to restrict Nodal
expression to the left side of the embryo to push for molecular asymmetry.

Lecture XXIII

16

T GF- S ign alin g: R ec ep to rs a nd C orecep to rs

Terminology
Term
Receptor Serine/Threonine
Kinase
Catalytic Receptor
Type I Receptors
Type II Receptors
Homodimer
Heterodimer
Constitutively Active
Induced Proximity Model
GS Region
SMAD Protein
Transcription
Phosphorylation
SMAD Signaling
Non-SMAD Signaling
Endoglin
Co-Receptor (Accessory
Receptor)
Endocytosis
Clathrin-coated Pit
Lipid-Raft
Transcription Factors

Definition
Type of kinase enzyme that phosphorylates the OH group of serine or
threonine, playing a role in regulation of cell proliferation, apoptosis,
differentiation, and embryonic development. TGF
Transmembrane receptor where the binding of an extracellular ligand causes
intracellular enzymatic activity.
Protein receptor in heteromeric complex which is the main switch involved in
the signal transduction.
Protein receptor in heteromeric complex which can remain active, but needs
a allosteric, conformational change in order to fully activate signal
transduction.
Macromolecular complex formed by two identical molecular subunits.
Macromolecular complex formed by two non-identical molecular subunits.
Type of activity in which the molecule is constant and active, but is not in
appropriate conformation to phosphorylate another receptor.
Ligand binding brings proteins together in the membrane and this close
proximity allows for signaling to occur.
Regulatory site for TGF- type I receptor. It needs to be phosphorylated by
TGF- type II receptor for it to occur.
Intracellular proteins that transduce extracellular signals from TGF- ligands
to the nucleus to activate downstream gene transcription. Exist in three
classes: (1) Receptor-regulated, (2) Common-mediator, and (3) Antagonistic
Production of a complementary RNA copy (mRNA) from a sequence of DNA.
Addition of a phosphate group to a protein or another organic molecule,
activating or deactivating protein enzymes.
Signaling involves transduction of extracellular signaling to the nucleus. They
form a trimer or two receptor-regulated SMADs and one co-SMAD to create a
transcription factor to regulate expression of certain genes.
Conveyance of non-SMAD signaling proteins such as p38 or MAPK, with no
use of SMAD proteins.
Type I membrane glycoprotein on cell surfaces that is part of the TGF-
receptor complex.
Cell surface receptor that binds a signaling molecule in addition to a primary
receptor to facilitate ligand recognition and initiate signaling.
Process by which cells absorb molecules through engulfment.
Involved in clathrin-mediated endocytosis, which is formed from the inward
budding of plasma membrane vesicles to allow internalization of the
molecules.
Organization of glycosphingolipids and protein receptors to compartmentalize
cellular processes by serving as centers for assembly of signaling molecules.
Protein that binds to specific DNA sequences to regulate transcription.

Lecture Objectives
Learn about the different types of receptors and co-receptors in the TGF- superfamily.
Type
Examples
I
TRI (ALK5), ActRIB (ALK4), ALK 7, ALK1, ALK2, ALK3, ALK6
II
TRII, ActRII, ActRIIB, BMPRII, MISRII
Generally there are two types of receptors: Type I and II. In the signaling pathway, be
sure to remember that Type II must be activated in order for activation of Type II to
occur. They are quite structurally similar, and can only be differentiated by peptide
mapping. They form homodimers and heterodimers. The number of ligands very
much exceeds the number of receptors, so there can be convergence on signaling,
particularly to a receptor. Consequently, there are combinatorial interactions, or
diverse responses by small numbers of receptors and communication. The
significance is simply because there is one way for cells to generate the capacity for
diverse responses derived from a small number of ligands and receptors (with TGF-
superfamily responding to a specific the TGF- receptor). It also allows for crosstalking
between different signals. However, cells need to respond appropriate to the signal.

17

Know how signal transduction occurs through the receptors.

The general signaling pathway to understand is that it is activated by
an extracellular ligand. The receptors involved in TGF- Signaling
typically involved Receptor Serine/Threonine Kinases, which are
consequently involved serine/threonine phosphorylation, which has
intrinsic catalytic activity. The pathway generally involves several
steps:
1. TGF- brings together two Type I Receptors with Type II
Receptors.
2. Type II Receptors phosphorylate and activate type I
receptors.
3. The R-SMAD proteins bind to Type I Receptors to be in
complex with SARA.
4. Type I receptor phosphorylates R-SMAD, promoting
dissociation from the kinase and SARA.
5. The product binds to a Co-SMAD.
6. SMAD hetero-oligomer enters the nucleus.
7. The SMADs associate with other DNA-binding proteins
to activate or inhibit transcription of specific genes.
The TGF- dimer binding results in a heterotetrameric complex
of receptor subunits. The receptos essentially cluster to form heterotetrameric complex, consisting of 4
receptors and 2 ligand molecules [4+2]. A jigsaw arrangement can explain the specificity of the TGF-
superfamily to the TGF- receptor. It is simply a stepwise, allosteric, cooperative mechanism. When the
Type II receptor binds to TGF, it allows a conformation that allows the Type I receptor to fit, yielding the
TGF- receptor complex. Essentially, the Type I receptor binds much more favorable in the presence of
another factor (Type II), because the receptor affinity is higher for Type I after Type II enters. This is not
necessary for other proteins such as BMP, which binding and activation of the complex can occur
simultaneously. The system is to bring the Type I and II receptors for an intimate embrace. The complex
puts the cytoplasmic kinase domains in a catalytically favorable orientation. This system is also associated
with the induced proximity model, in which the ligand binding causes proteins to move closer together in
the membrane and this close proximity causes the signaling to occur. The phosphorylation of the GS
region of the TGF- 1 receptor activates the kinase. The GS region is the regulatory factor that interferes
with the kinase. The GS region will move out of the way when the serines of the Type I receptor are
phosphorylated. Type II does not have the GS, but Type I needs to move the GS region to cause the
desired activation. Phosphorylation allow allows the type I receptor to phosphorylate SMAD proteins, and
the phosphorylation of the SMAD proteins allow SMADs to accumulate in the nucleus. Phosphorylation is
used both to activate Type I receptors and SMAD proteins, and thus one can conclude that phosphorylation
is the mechanism to activate or deactive proteins or recruit proteins to a complex.
The%
TGF9[beta]%
familyVolume%
50%
of%
Cold%
Spring%
Harbor%
monograph%
series%
(CSHL%
Press,%
2008),%
pp.%
1114.

Endoglin has been noted as a co-receptor that is abundant in the vascular endothelial cells, mainly to
potentiate signaling through the Type I and Type II receptors, but signaling can occur without the coreceptor. Endoglins function still remains a clinical mystery, but co-receptors play a role in endocytosis
(especially in clathrin-mediated uptake) of TGF- receptors, and the signaling may be ongoing.
Know the difference between SMAD and non-SMAD signaling via the TGF- pathway.
TGF- signaling typically involves SMAD-mediated responses, but non-SMAD responses have also been
noted. Some receptors act in other ways without utilizing SMAD, and was discovered through observation
of rapid effects that were not involving SMAD proteins. This may allow for a bypass mechanism to allow
for a desired effect in the event of faulty SMAD signaling.
Discuss the parameters that determine what kind of response is
elicited in cells during signaling.
Now this leads to another question: what determines what kind of response
will be elicited by TGF- superfamily ligands in any particular cell?
Experiments and studies have lead to these factors:
1. Ligands
2. Receptors and Co-Receptors
3. SMADs
4. Cell type-specific cooperating transcription factors

Lecture XXIV
Trans cr ip tion al Reg ulatio n in Physiolog y

18

Terminology
Term
Central Dogma of
Molecular Biology
Differential Expression
Messenger RNA (mRNA)
Ribosomal RNA (rRNA)
Transfer RNA (tRNA)
Small, noncoding RNAs
Transcription Unit
Transcriptional Control
Acute Regulation
Long-Term Regulation
RNA Polymerases (I, II, III)

Transcription Cycle
Initiation Phase
Elongation Phase
Termination Phase
Gene Promoter
TATA Box
Initiator Element
Basal Promoter
Preinitiation Complex
General Transcription
Factors
(TATA Box Binding
Protein ) TBP
Closed Complex
Open Complex

Conformational Change
Promoter Proximal
Elements
Enhancer Elements
Gene-Specific

Definition
Framework of comprehsnion in the transfer of information between DNA,
RNA, and protein among living organisms, in which DNA can yield RNA, which
then yields protein. However, this does not happen in the reverse,
generating a one-way flow of information.
Differences among cell expression even though the cells have the same DNA,
mainly because they express different parts of the DNA.
Molecule of RNA that encodes information for the protein product.
RNA component of the ribosome, providing the mechanism for decoding
mRNA into amino acids and interacts with tRNAs during translation.
Adaptor molecule composed of RNA utilized in bridging genetic code in
mRNA.
Short ribonucleic acid molecule in eukaryotes that are regulatory involved in
post-transcriptional regulation.
Stretch of DNA transcribed into an RNA molecule to allow for eventual
translation to protein.
Regulation of gene expression by controlling number of RNA transcripts of a
region of DNA. Major regulatory mechanism of protein synthesis.
Processes that need to be regulated in the range of seconds to minutes
involving changes in protein activity, brecause these changes can occur
rapidly.
Processes that are regulated over a longer time period typically involving
transcriptional regulation.
Enzyme that produces RNA. There are three types:
RNA Polymerase I: Involved in the transcription of DNA to rRNA
RNA Polymerase II: Involved in the transcription of DNA to mRNA or
snRNA.
RNA Polymerase III: Involved in the transcription of DNA to tRN, 5S
rRNA, or 7S RNA of signal recognition particle.
Process by which the DNA transcribes an RNA product. Contains three
phases: (1) Initiation, (2) Elongation, and (3) Termination
Phase of transcription where promoters designate area and allow binding of
RNA polymerase to DNA promoter.
Phase of transcription where DNA template strands allows for the synthesis of
an RNA copy.
Phase of transcription consisting of the halting of RNA synthesis and release
of mRNA.
Region of DNA that facilitates transcription of a gene.
DNA sequence found in promoter region of genes. Part of the promoter
sequence, it is the binding site of general transcription factors and involved in
transcription.
DNA sequence element that overlaps a transcription start site and allow
determination of start site location in a promoter and enhancing strength of a
promoter with a TATA box.
Promoter elements that can direct low levels of transcription, but are
insufficient for full expression of a gene.
Large complex of proteins necessary for transcription of protein-coding
genes.
Transcription factors involved in the transcription of class II genes to mRNA
templates.
General transcription factor that binds to TATA box.
Complex state of a protein where the aqueous environment is isolated with
the non-aqueous.
Complex state of a protein where the protein opens, allowing for aqueous and
non-aqueous environment to interact. In this case, the open complex allows
the transcription start site to be unpaired and allows exposure to the
template strand to allow nucleotides to join.
Change in the proteins structure in response to the environment or other
factors.
Short regions of DNa involved for constitutive expression, or regulated
expression.
Short region of DNA that can be bound with proteins to enhance transcription
levels of genes, which work by increasing the rate of initiation from a basal
promoter.
Diverse protein involved in gene regulation that are specific to a seuqnece of

19

Transcription Factors
Chromatin Structure

5 to 3 Direction
Pausing and Editing
mRNA Capping
Poly-A Tail
mRNA Export
Nuclear Pore Complex

DNA.
Combination of DNA nad proteins that make up the contents of the nucleus of
a cell. Its function is to package DNA to a smaller volume to fit into the cell
and to strengthen DNA to allow cell division and control gene expression and
DNA replication.
Chemical orientation of a strand of nucleic acid, with the start designated as
5 and the end as 3.
Process in elongation phase consisting of halting transcription to allow
replacement or removal of nucleic acid.
With the help of enzymes, a process in which a cap is added to the 5 end of
the nascent transcript, allowing protection against degradation and to
promote translation.
String of adenines added to the 3 end of the transcript to designate end of
sequence.
Release of mRNA from nucleus through nuclear pore complex.
Pore in the nucleus in which the nascent strand of mRNA is release into to
arrive at the cytoplasm.

Lecture Objectives
Know the difference between transcriptional and non-transcriptional regulation of gene
expression.
In order to understand transcriptional and non-transcriptional regulation of gene expression, we need to
first understand the elements of transcription. Transcription is simply the generation of mRNA from DNA,
which (in eukaryotes) occurs in the nucleus. It is one of the processes within the Central Dogma, in which
DNA synthesizes RNA, and RNA consequently produces a protein. This one-way flow of information is key
to the biological processes at a unicellular and multicellular level. From this, researchers eventually
discovered mRNA, tRNA, rRNA, miRNA, and snRNA. All cells have the same genes (as ingrained in the form
of DNA), but the cells are specialized due to the expression of different sets of genes.
Expression of genes is different in each type of cell. Some are on all the time (constitutively), or only on at
certain times. It can be also expressed in a cell-type specific manner. At times, some are expressed at
high levels, while other are expressed at lower levels, and even some are expressed at higher levels only
when specific cell surface receptors or cytoplasmic receptors are activated by ligand binding.
Now, to discuss the genetic material, there are two major types of genetic material: DNA and RNA. DNA is
a double helix with deoxyribose sugars consisting of one side with a strand of nucleotides while the other
has a strand of the complementary sequence, according to Watson-Crick base pairing. RNA is a singlestranded molecule with a ribose sugar and contains uracil instead of the thymine in DNA. There are
several types of RNA, but only one type of DNA. The types of RNA are listed at the table below:
Type (Abbreviation)
Description
% Of
RNA
Messenger RNA (mRNA)
Major encoding element for proteins.
< 10
Ribosomal RNA (rRNA)
Major component of ribosomes.
75
Small Stable RNAs
Encompass RNAs that are mainly adaptor proteins.
15
Transfer RNAs (tRNAs) are involved in protein
synthesis.
Small Nuclear RNAs (snRNAs) are involved in RNA
splicing, regulation of transcription
Small noncoding (ncRNAs)
Regulatory RNAs involved in controlling gene expression.
Small.
or microRNAs (miRNA)
The strand of DNA that is being transcribed is typically referred to
as the transcription unit. After transcription occurs, the mRNA is
produced, but undergoes editing and splicing (to remove introns
will keeping the exons at certain splice sites) to yield the mature
mRNA. Transcriptional control is especially crucial to prevent
expression of possible mutations. Transcriptional regulation is
altering the rate of gene expression by altering the rate of
transcription. Non-transcriptional regulation is associated with the
rate of gene expression by altering the protein activity, typically
after mRNA has been synthesized. For example, non-SMAD
mediated responses dont involve changes at the level of transcription, so it can be considered a nontranscription regulation.
Figure 7-5 Molecular Biology of the Cell ( Garland Science 2008)

20

Learn about acute responses versus longer-term responses.

Acute regulation typically encompass processes that need to be regulated in the range of seconds to
minutes that involve changes in the protein activity, because these changes that can occur rapidly.
Altering the environment of the protein to chemical phosphorylation of isomerization can affect the
proteins activity. Long-term regulation involves processes that are regulated over a longer time period,
typically involving transcriptional regulation. There are several advantages of this. One, it is energetically
efficient, only producing a protein when it is needed. Second, it allows the cell to switch expression of one
type of gene to another, particularly during development or differentiation. Finally, it can also do largescale expression of a whole network of genes involved in a particular pathway or process.
Know the basics about the basal transcription
machinery. Learn about TFIID, TBP, and TATA
boxes.
In order to understand the machinery, it is necessary to
comprehend the various parts involved. Recall that
RNA polymerase is involved in the synthesis of RNA
from DNA. However, there are three types of RNA.
RNA polymerase I is involved in the synthesis rRNA,
and RNA polymerase III is involved in the production
tRNA, 5S rRNA, and 7S RNA of signal recognition
particle. RNA Polymerase II is the enzyme of interest for transcription, which yields mRNAs and some
snRNAs. RNA Polymerase II is a large multiprotein complex with a repeptive carboxylterminal domain. The conserved portions of the residues are located in the inner
surfaces of the enzymes. The structure of the enzyme contains a cleft for the DNA and
asite for nucleotides to enter, as well as a channel for newly synthesized RNA to exit.

Know the transcription cycle.

The transcription cycle is a process creating RNA from DNA. It contains three steps:
(1) Initiation, (2) Elongation, and (3) Termination. Among these steps (which are all
regulated), initiation is the most regulated step. Prior to Initiation, a pre-initiation

complex
must be formed because RNA polymerase II on its own cannot initiate
transcription from promoters. Consequently, factors, known as general transcription
factors, are required to form a complex. For RNA Pol II, there are more than 20
proteins. Though there are other factos involved in this complex, the first one to go in
is transcription factor IID (TFIID). The binding of TBP to the TATA box leads to a
pronounced bend in the DNA and recruitment of other factors.

Initiation of transcription (the Initiation Phase) involves a shift of the RNA Polymerase/DNA
Complex from a closed complex to an open complex conformation. This conformation

change
in the polymerase causes a region of DNA around the transcription start site to
become unpaired, allowing exposure of the template strand. Consequently the first
ribonucleotides are joined. At this point, several sequences, such as promoters, proximal
promoter elements, and enhancers, are also add to spur effects on the rate of initiation
from the basal promoter.

The You'are'
Elongation Phase involves the clearance of promoter that needs to occur before synthesis begins.
terminated!'
The
transcript is extended in a 5 to 3 direction at a rate of approximately 30-100 nucleotides per second
Termina1on'Phase:'As'
by
the following chemical reaction:
RNA'Pol'II'reaches'the'

(NMP)n+ NTP (NMP)n +1+ PP i

. During this process,

end'of'a'gene'(3'end),'
pausing
and editing may occur. As the new (or nascent) transcript emerges, a special cap structure is
the'RNA'gets'cleaved'at'
the'polyadenyla1on'site'
added
on to the 5 end. This cap help to protect against degradation and later will promote translation of
and'a'poly=
A'tail'as'added'
the
message
in the cytoplasm.
to'the'transcript.''The'
polymerase'may'con1nue'
transcribing'for'a'while'
Termination
of transcription (the Termination Phase) involves
but'soon'falls'off.'
II reaching the end of the gene at the 3 end. Once this
gets cleaved at the polyadenylation site and a poly-A tail is
transcript. The polymerase may continue transcribing for a
falls off. After splicing occurs, the mature mRNAs are
the nucleus through the nuclear pore complex.

Figure 6-38 (part 2 of 3) Molecular Biology of the Cell ( Garland Science 2008)

RNA Polymerase
occurs, the RNA

added to the
while but soon
exported from

Be aware of the role of promoters, proximal promoter

elements, and
enhancers.
Promoters are the sum of DNA sequences necessary for
transcription
initiation. Other elements include a TATA box and initiator
element, which
is found near the start site of transcription, and consequently
part of the basal
promoter. Basal promoter elements can direct low levels of transcription, but are insufficient for full
expression of a gene, so (as a result) promoters also contain promoter proximal elements that are needed
for constitutive expression or regulated expression. Another group of sequences, known as enhancers,
typically increase the rate of initiation from a basal promoter. They can be distant from the start site, and

21

exhibit flexibility (allowing itself to work in different positions or orientations), and contain clusters of
regulatory elements. Such regulation can have an effect on gene-specific transcription factors as well as
chromatin structure. The following table summarizes promoters, proximal promoter elements, and
enhancers.
Sequence
Promoter

Proximal Promoter
Elements

Diagram

Description
Loosely defined as the sum of DNA sequences
necessary for transcription initiation. TATA box and
initiator element are found near start of transcription
as part of a basal promoter.
Needed for constitutive/regulated expression.

Enhancers

Flexible part of the sequence that can be far from

the start site that increase the rate of initiation from
a basal promoter. They can contain clusters of
regulatory elements.

Lecture XXV
S tr ateg ies fo r Tra nsc riptio na l Regu la tio n in Eu ka ryotes

Terminology
Term
Promoter proximal
elements
Enhancer Elements
Insulator Elements
Locus Control Regions
Gene Specific
Transcription Factors
Major Groove

Hydrogen Bonding
Combinatorial Control
Synergy
Cooperativity
Mediator Complex
Lambda Repressor

DNA Binding Domain

Activation Domain
Repressors
Competition
Masking

Definition
Proximal sequence upstream of the gene that tends to contain primary
regulatory elements.
Elements that increase the rate of initiation from a basal promoter, which can
be distant from start site and contain clusters of regulatory elements.
Genetic boundary eleent that is an enhancer-blocking element or a barrier
against condensed chromatin proteins spreading onto active chromatin.
Regions defined by their ability to enhance the expression of linked genes.
Binding sites for proteins allow differential control of gene transcription.
Type of groove seen in DNA structure containing the nitrogen and oxygen
atoms of the base pairs pointing inward toward the helical axis. It is more
dependent on base composition and may be the site for protein recognition of
specific DNA sequences or regions.
Attractive interaction of a hydrogen atom with an electronegative atom.
Complex regulatory regions (in enhancers and promoters) are constructed
from different combinations of simple regulatory molecules.
Two or more elements functioning together to produce a result not
independent obtainable.
Behavior observed in enzymes and receptors that have multiple binding sites
where the affinity of the binding sites for a ligand is increased or decreased
as a consequence of binding of the ligand to the receptor.
Multiprotein complex that functions as a transcriptional coactivator.
Switch in the lifecycle of a bacteriophage responsible for maintenance of
lambda phage. It binds to operator associated with the RNA polymerase
promoter to prevent RNA polymerase from initiating transcription, and cannot
enter the lytic cycle.
Independently folded protein domain that contains at least one motif that
recognizes DNA.
Protein domains involved in the formation and stability of the preinitiation
complex, activating transcription.
DNA-binding protein that regulates expression of one or more genes by
binding to the operator and blocking attachment of RNA polymerase to the
promoter, blocking transcription.
Method of utilizing transcription factors as repressors by competitive DNA
binding.
Method of utilizing transcription factors as repressors by masking the

22

De Novo Synthesis
Coactivator
Histone Acetyltransferase
Corepressor
Gene Silencing
DNA Methylation
Histone Methylation
Genetic Program
Network Motifs

activation surface.
Synthesis of complex molecules from simple molcules.
Protein that increases gene expression by binding to an activator containing
the DNA binding domain. Coactivators cannot bind DNA by itself.
Enzymes that acetylate conserved lysine amino acids on histone proteins.
Substance that inhibits the expression of genes, by indirect means
(interaction with repressor proteins that in turn bind to the promoter)
The deactivation of a gene by a mechanism other than genetic modification,
meaning that it would be expressed under normal circumstances.
Addition of a methyl group to the 5 position of the cytosine pyrimidine ring or
the number 6 nitrogen of the adenine purine ring on DNA.
Modification of certain amino acids in a histone protein by addition of one,
two, or three methyl groups.
Physiological change brought about by a temporal pattern of activation of a
particular subset of genes.
Connectivity patterns that occur more often in comparison to random
networks.

Lecture Objectives
Know the different types of DNA regulatory elements that control transcription of a gene.
Regulator
Illustration
Function
Characteristics
y
Element
Promoter
Proximal sequence
Can be within 300 base pairs
Proximal
upstream of the gene that
upstream or downstream of the basal
tends to contain primary
promoters.
regulatory elements.
Enhancer
Elements that increase the
Can be distances away from the start
rate of initiation from a
site. They will work in different
basal promoter, which can
positions or orientations. They also
be distant from start site
contain clusters of regulatory
and contain clusters of
elements.
regulatory elements.

Insulator'Elements'

Insulator

Insulators'protect'regions'of'a'chromosome'from'the'
effects'of'neighboring'regions.''

Locus'control'regions'

Figure 7-62 Molecular Biology of the Cell ( Garland Science 2008)

Locus
Control
Regions

Genetic boundary element

that is an enhancerblocking element or a
barrier against condensed
chromatin proteins
spreading onto active
chromatin.
Regions defined by their
ability to enhance the
expression of linked
genes.

Figure 7-61 Molecular Biology of the Cell ( Garland Science 2008)

Insulators are involved in the

protections of regions of a
chromosome from the effects of
neighboring regions. They suppress
activation of one gene, so that the
enhancer only affects a specific gene.
Locus control regions are short
regions of DNA rich in binding sites
for transcription regulators, which
create open chromatin promoting
the expression of nearby genes.
These regions can be important in
regulating the expression of a cluster
of nearby genes, so that they are
expressed in the correct order during
development or in the current
location in the embryo. All these
regulatory elements contain binding
sites for proteins called gene-specific
transcription factors.

It is important to remember that all cells have the same genes in the form of DNA, but cells are specialized
because of the differences in expression of different sets of genes. It can also respond to its environment
by turning on or off specific genes. Transcription initiation is key. A host of general transcription factors
are required to form the preinitiation complex. Basal promoters only allow for basal levels of expression.
Anything that promotes the formation of the preinitiation complex or stabilizes it will elevate transcription
of the gene. It yields contact-mediated effects of gene-specific transcription factors and effets on

23

chromatin structure, which alter DNA accessibility for other transcription factors. There are four types of
regulatory elements: (1) promoter proximal elements, (2) enhancer elements, (3) insulator elements, and
(4) locus control regions. Promoter proximal elements are sequences that are upstream or downstream in
proximity of the basal promoter (landmarked by TATA box). It has all the binding sites upstream of the
promoter, within the first 300 base pairs, but must be near the transcription site. They are identified by a
reporter gene assay, with the promoter gene being attached the reporter gene. Enhancers are elements
that increase the rate of initiation from a basal promoter. They can be great distances away from the start
site, maintain flexibility, and contain clusters of regulatory elements. Insulators are involved in the
protections of regions of a chromosome from the effects of neighboring regions. They suppress activation
of one gene, so that the enhancer only affects a specific gene. Locus control regions are short regions of
DNA rich in binding sites for transcription regulators, which create open chromatin promoting the
expression of nearby genes. These regions can be important in regulating the expression of a cluster of
nearby genes, so that they are expressed in the correct order during development or in the current
location in the embryo. All these regulatory elements contain binding sites for proteins called genespecific transcription factors.
Learn about gene-specific transcription factors.
Remember that all the regulatory elements contain binding sites for proteins
called gene-specific transcription factors. They make up approximately 6-8% of
human genes. They are critical for a diverse array of cellular processes.
There are about 1,400 transcription factors out of 20,000-25,000 genes.
Transcription factors recognize and bind to specific DNA sequences, such as
homeodomains, zinc fingers, glucocorticoid receptors, and leucine zippers.
Most factors recognize exposed chemical groups in the major groove of the
DNA double helix. The type of bonding involved is hydrogen bonding. The
specificity (to regulate only the right genes) comes binding sites. If the factor
forms a dimer with itself or another protein, this will increase the specificity
because it requires two adjacent sites to be present. Different dimer pairs
can generate the novel binding sites.
Be familiar with the concepts of combinatorial control and synergy with
regard to transcription factors.
Combinatorial control is when complex regulatory regions (in enhancers and
promoters) are constructed from different combinations of simply regulatory
modules. It allows for the integration of multiple regulatory signals by cells and
for very complex spatial patterns of gene expression. In certain positions along a
developing embryo, certain genes are enhanced and suppressed (and the
concentrations of the regulatory proteins increase or decrease) along certain
position of the embryo.

SFcky#
surfaces#
that#
could#
interact#
with#
TranscripFon#
factors#
bind#
to#
the#
DNA,#
but#
how#
several#
protein#
surfaces#
do#
they#
acFvate#
or#
repress#
transcripFon?#

Transcription factors can work synergistically. Transcription with one protein can

increase the number of regulatory proteins in transcription. Synergy is achieved

in two ways: (1) classical cooperativity involves the binding of one factor makes it
easier for the other to bind, or (2) two factors may be better able to recruit a
coactivator by touching different parts of it. Binding of one factor may result in an
Cells'regulate'transcrip4on'factors'in'several'
alteration in chromatin structure so that another binding site becomes more accessible. By#
The
functions
of
affecFng#
the#
formaFon#
or#
stability#
of#
the#
ways,'depending'on'the'factor'and'pathway'
PreiniFaFon#
Complex#
synergy is to allow a sensitive switch to turn genes on or off. The DNA binding
specificity increases. The integration of different signals is possible. Cooperative
binding can affect the life of the various cells.
How#
does#
this#
relate#
Know the basics about how transcription factors activate or repress
transcription.
Transcription factors bind to the DNA, but can directly or indirectly activate or suppress
transcription by affecting the formation or stability of the preinitiation complex. They
have modular activation domains and DNA binding domains. The activation domains
have different regions: acidic, proline-rich, and glutamin-rich regions. They are, simply put, stick surfaces
that could interact with several protein surfaces. These transcription factors can be repressors also,
through (1) competition (competitive DNA binding), (2) masking (masking the activation surface, or (3)
direct interactions with general factors.

24

to#
signal#
transducFon#
pathways#
in#
the#
cell?#

Pollard'Fig.'15D21'

Know the different ways that transcription factors can be

regulated.
Cells regulate transcription factors in several ways, depending on the
factor and pathway. There are several ways to regulate transcription
factors: (1) de novo synthesis, (2) ligand bindng, (3) phosphorylation, (4)
heterodimer formation, (5) dimer dissociation, and (6) subcellular
localization.
Know that transcription factors can also work by affecting
chromatin structure.
Transcription factors can also exert their effects by altering chromatin
structure rather than by direct contact-mediated effects on the preinitiation complex. The activating factor may recruit a coactivator that is
a histone acetyltransferase. The repressor may recruit a corepressor that
is a histone acetylase. Acetylation of histone tails loosens up the
chromatin structure.

Transcrip4on'factors'can'also'exert'their'effects'by'
altering'chroma4n'structure'rather'than'by'direct'
contactDmediated'effects'on'the'preDini4a4on'complex'
Pollard'Fig.'15D20'

Ac4va4ng'factor'may'
recruit'a'coac4vator'that'
is'a'histone'
acetyltransferase.'

Lecture XXVI

Repressor'may'recruit'a'
corepressor'that'is'a'
histone'deacetylase'

Ro le o f SM ADS in Tra ns cr ip tion al Reg ulatio n via the TG F-

Pathway

AcetylaFon#
of#
histone#
tails#
loosens#
up#
chromaFn#
structure#

Terminology
Term
Nuclear Pore Complexes
MH1 Domain
MH2 Domain
Linker Region
Receptor activated SMAD
(R-SMAD)
Common Mediator SMAD
(Co-SMAD)
Inhibitory SMAD (I-SMAD)
SMAD Box
-Hairpin Loop

L3 Loop
-Helix
Major Groove of DNA
Gel Shift Assay

DNA Binding Motif

Sequence-Specific
Transcription Factors
Coactivators

Definition
Large protein complexes that cross the nuclear envelope.
Domain in SMAD proteins that is involved in DNA binding and interaction with
transcription factors.
Domain in SMAD proteins that are involved in receptor interaction, Smad
oligomerization, transcriptional activation, interaction with CBP/p300, and
interaction with transcription factors.
A short synthetic double-stranded DNA that is usually ligated to doublestranded DNA to introduce restriction sites or sequence tags.
Transcription factors that transduce extracellular TGF- ligand signaling from
cell membrane bound TGF- receptors into the nucleus where they activate
transcription TGF- target genes. Includes SMAD1, SMAD2, SMAD3, SMAD5,
and SMAD8/9.
Transcription factor that interacts with R-SMADs to participate in signaling.
Includes only SMAD4.
Type of SMAd involved in the modulation of TGF- ligands, which includes
SMAD6 and SMAD7. They inhibit transcription.
Site where -hairpin loop makes H-bonds with unpaired groups, which allows
binding in a sequence-specific manner. The sequence of the box is GTCT.
Simplest structural motif involving two strands that look like a hairpin,
which consists of two strands that are adjacent in primary structure oriented
in an antiparallel arrangement (where the N-terminus of one sheet is adjacent
to the C-terminus of the next).
Region of MH2 domain that actually interacts with the Type I receptors. Small
sequence differences along the L3 loop residues allow determination of the
right structure.
Common motif in the secondary structure of protiens containing a righthanded coil or spiral conformation.
Wider region of the DNA helix
Common affinity electrophoresis technique used to study protein-DNA or
protein-RNA interactions. This can determine if a protein or mixture of
proteins is capable of binding to a given DNA or RNA sequence, and ca
indicate if more than one protein is involved in the binding complex.
Protein domain that contains at least one motif that recognizes double or
single-straned DNA. It can recognize a specific DNA sequence or have a
general affinity to DNA.
Transcription factors that bind to specific sequences along DNA, which allow
for a coding system for transcription.
Protein that increases gene expression by binding to an activator, which
contains a DNA binding domain.

25

Corepressors
Target Genes
Sequential and SelfModifying Signaling
Feed-Forward Loop

Network Motif
Gene Regulatory Network
Post-Translational
Modification
Phosphorylation
Mitogen Activated Protein
(MAP) Kinases
Dephosphorylation
SMAD Phosphatases
Ubiquitination
Ubiquitin Ligases
Crosstalk
Subcellular Localization

Substance that inhibits the expression of genes, in which is downregulates

the expression of genes not by direct interaction with a gene promoter but
indirectly through interaction of repressor proteins that bind to the promoter.
Gene encoding the regulated product, including any linked regulatory
elements and selectable markers.
Methods of signaling in which the signaling may induce the expression of
another transcription factor that can then cooperate with the signaling to
regulate other targets.
Pathway within a control system that passes a controlling signal from the
source in the control systems external environment to a load elsewhere in its
external environment. This allows the cell to be sensitive to the duration of a
signal.
Connectivity-patterns that occur much more often than they do in random
networks.
Collection of DNA segments in a cell that interact with each other indirectly
and with other substances in the cell, governing the rates at which genes in
the network are transcribed into mRNA.
Chemical modification of a protein after its translation.
Addition of a phosphate group to a protein or other organic molecule.
Serine/threonine-specific protein kinase that respond to extracellular stimuli
and regulate various cellular activities.
Removal of phosphate groups from an organic compound by hydrolysis.
Enzyme that regulates the removal of phosphate groups from active SMAD
proteins.
Enzymatic, protein post-translational modification process in which ubiquitin
is added to the protein. It targets SMAD for degradation by the proteasome.
Protein that in combination with an E2 enzyme that causes the attachment of
ubiquitin. It is involved in polyubiquitination, marking proteins for
degradation by the proteasome.
Instances in which one or more components of a signal transduction pathway
affect a different pathway.
Subdivisions of a eukaryotic cell that allow functionally distinct membrane
The'distribu8on'of'SMADs'between'cytoplasm'
bound compartments.
and'nucleus'is'altered'during'signaling'

Lecture Objectives
Know the role of SMADs in the TGF- family-signaling
pathway.
Remember that anything that promotes or stabilizes the preinitiation
complex will activate transcription, and this is the same for the
opposite. Transcription factors bind to specific DNA sites and act
synergistically with other factors to achieve combinatorial control.
Consquently, transcription factors can be regulated, and whether or not a
gene is transcribed depends on what regulatory elements it has, what
transcription factors are around, and how accessible is the DNA.
Transcription factors work by contact-mediated effects or by altering
chromatin structure. Thus transcription factors have modular activation
domains and DNA binding domains. They have different types: (1) acidic
regions, (2) proline-rich regions, and (3) glutamine-rich regions. These
are essentially surfaces that could interact with several protein surfaces.
It should also be remembered that transcription factors can also act as
repressors also, either by competition, masking, or direct interactions.
Cells regulate these transcription factors in several ways, depending on
the factor and pathway.
The ligand/receptor complex puts the cytoplasmic kinase domains in
a catalytically favorable orientation. The Type II receptor
phosphorylates the Type I receptor. The phosphorylation of the GS
region of the TGF- type I receptor activates this kinase. This cannot
happen in Type II receptors because the Type II receptors do not
have this region. The phosphorylation of the Type I receptor
activates it to phosphorylate SMAD proteins.
The SMADs are the gene-specific transcription factors that are used
to regulate target genes affected by TGF- family ligands. The

26

B.'Schmierer,'C.'S.'Hill,'Nat+
Rev+
Mol+
Cell+
Biol+
8,#970&
82#(2007).'

Ac8ve'SMADs'
accumulate'in'
the'nucleus'

SMADs will bind to specific DNA sites. Type I receptors activate different SMADs, and the distribution of
SMADs between cytoplasm and nucleus is altered during signaling. When ligand binds, there is a steady
state shift towards the accumulation in the nucleus, and
difficulty to export out active SMADs. Consequently, there
is an accumulation of the SMADs in the nucleus.
SMADs enter and exit the nucleus through nuclear pore
complexes. The R-SMADs are associated in the cytoplasm
with SMAD Anchor for Receptor Activation (SARA). Thus,
SMADs can either be recycled (by dephosphorylation) or
degraded (by ubiquination).
Learn about the different classes of SMADs
(Receptor activated, co-SMAD, Inhibitory SMAD).
All in all there are 8 SMADS, which are divided into three
types: (1) receptor activated (R-SMAD), (2) common
mediator SMAD (co-SMAD), and (3) inhibitory (I-SMAD).
The R-SMADs are involved in the activation of transcription
upon ligand binding. Co-SMADs do not get phosphorylated, and allow for increasing the affinity of the
binding of the DNA to the R-SMAD. I-SMADs are involved in the inactivation of repression. They lack MH1
domains.
Development+
136,#3699&
714#(2009).

BeSMAD'proteins'consist'of'MH1'and'MH2'
familiar with the different functional domains of SMADs (MH1, MH2, linker region, -hairpin
loop, L3domains'and'a'linker'region'
loop).
SMAD proteins consist of MH1 and MH2 domains and a linker
MH1' Linker' MH2'
TGF?'type'I'
region. MH1 is going to bind to the SMAD box, and allows
receptor'
receptor specificity to binding. MH2 is phosphorylated by the
Phosphoryla8on'site'
cytoplasmic'
Type I receptor. The MH2 domain contains the L3 loop region that
domain'
actually interacts with the Type I receptors. The L3 loop is
SMAD'
responsible for the specificity of the SMAD to the DNA binding
site. The small sequence differences of the L3 loop residues can
Specificity'
determine whether the protein is in the right structure. MH1
DNA'
domains are involved in DNA binding and interaction with
transcription factors. The MH2 domain is involved in receptor
activation, SMAD oligomerization, transcriptional activation,
interaction with CBP/p300, and interaction with transcription factors. The phosphorylation will occur at the
candidate target sites. The linker region is a short, synthetic strand of DNA that allows introduction of
restriction and binding sites.
'J.'Massagu,'Nature+
Reviews+
Molecular+
Cell+
Biology+
1,#169&
78#(2000).'

SMADs bind DNA through a secondary structure known as the -hairpin loop. They bind to the SMAD box,
which has the sequence GTCT, and makes hydrogen bonds with unpaired groups, allowing binding in a
sequence-specific manner.
One can be able to demonstrate the presence of the binding site for a particular protein by a gel shift
assay. An individual can take a DNA sequence with the SMAD box and label fragment and run a gel
electrophoresis. The bound complex cannot move as easily as separated proteins. At high enough
concentrations, the binding sites will then be occupied. The DNA binding domainQuan8ta8ve'effects:''The'type'of'coopera8ng'
sequences are conserved
transcrip8on'factor'can'also'affect'the'threshold'of'
(
in different SMADs, and
especially
in all receptor
SMAD?mediated'responses.'
activated SMADs.
Know the basics about how
SMADs cooperate with other
transcription factors to
regulate specific target
genes.
Different SMADs have specific
effects in which the R-SMADSMAD4 complexes often
cooperate with other sequence-specific transcription factors. The high
affinity binding of SMADs typically requires cooperation with other factors.
Transcriptional complexes involving SMADs involve repetition of the recognition site or a SMAD box next to
a site next to a binding site. Different target genes are affected depending on the combination of SMAD
and cooperating transcription factor (cofactor). A lot of cooperative transcription factors might be cellspecific. Such cooperation has also quantitative effects. The type of cooperating transcription factor can
also affect the threshold of SMAD-mediated responses. A target gene can be either on or off at low signal
intensity depending on the cooperating transcription factor. It is also important in sequential and selfmodifying signaling. SMAD signaling may induce the expression of another transcription factor that can
then cooperate with the SMAD to regulate other targets. This is exemplary of a feed-forward loop. It is
also a time-dependent loop because it takes time to develop. A feed-forward loop allows the cell to be
B.'Schmierer,'C.'S.'Hill,'Nat+
Rev+
Mol+
Cell+
Biol+
8,#970&
82#(2007).'

27

In'this'example,'
the'target'gene'
is'either'on'or'
off' at'low'signal'
intensity)'
depending'on'
whether'the'
coopera8ng'
transcrip8on'
factor'is'X'or'Y'

sensitive to the duration of a signal. These kinds of gene network motifs are important to make a complex
system work. MH1 and MH2 domains are important for cooperativity. The differences in the amino acid
sequence in the MH1 and MH2 domains of the SMAd family members will determine with which
transcription factors they can interact. The linker region is considered the target for regulation via posttranslational modification, which allows for biological cross These'kinds'of'gene'network'mo8fs'are'
talk.

important'to'make'a'complex'system'work'

Know how SMADs are regulated.

SMADs can receive regulatory input from other signaling pathways through posttranslational modification in the following manner: (1) phosphorylation, (2)
dephosphorylation, and (3) ubiquitination. Phosphorylation can occur at various sites
by kinases other than Type I receptors. For example, phosphorylation by Erk MAP
kinase can impair nuclear translocation of the SMAD complex, while phosphorylation
by P38 MAP kinase or JNK can enhance SMAD activity. Because SMADs can be
phosphorylated by other kinases, crosstalk can occur between the TGF-/SMAD
pathway and other signaling pathways, such as those induced by growth factors.
Dephosphorylation occurs by SMAD phosphatases, which is one way to terminate
SMAD signaling. Ubiquitination is the last way, utilizingIn'addi8on'to'post?
ubiquitin ligases
to target
transla8onal'modifica8on,'another'
SMAD for degradation by the proteosome. In addition avenue'for'regulatory'input'are'Inhibitory'SMADs,'which'
to post-translational
modification, another avenue for regulatory input are inhibitory SMADs, which inhibit
inhibit'R?SMAD'ac8va8on'
R-SMAd activation. SMAD6 and SMAD7 are
Growth'Biology of theGrowth'
Figure 7-71 Molecular Biology of the Cell ( Garland Science 2008)
Figure 7-69 Molecular
Cell ( Garland Science 2008)
TGF?'
factor'
factor'R'
inhibitory SMADs, and act either by competing for
SMAD'6'and'SMAD'7'
binding to Type I receptor and preventing
are'inhibitory'SMADs'
Because'SMADS'can'be'
phosphorylation, or by binding to SMAD 4, which
phosphorylated'by'these'
ultimately prevents R-SMAD binding. Inhibitory
other'kinases,'crosstalk'
input can come from cytokine and growth factor
can'occur'between'the'
Act'either'by'compe8ng'
TGF?/SMAD'pathway'
signaling. Several signaling pathways can induce
MAP'kinase'
for'binding'to'Type'I'
and'other'signaling'
SMAD 6 and SMAD 7 gene expression. These
receptor'and'preven8ng'
pathways,'such'as'those'
SMADs can then inhibit TGF- signaling.
phosphoryla8on,'or'by'
induced'by'growth'
factors'(e.g.'mitogen?
ac8vated'protein'(MAP)
kinase'pathways)'

binding'to'SMAD'4'
(preven8ng'R?SMAD'
binding)''

Lecture XXVII
R.'Derynck,'Y.'E.'Zhang,'Nature+
425,#577&
84#(2003).'

A Lo ok a t S ome O th er Sig na ling S ys tems

that Aff ec t Tra ns cr ip tion

J.'Massagu,'Nature+
Reviews+
Molecular+
Cell+
Biology+
1,#169&
78#(2000).'

Terminology
Term
Cytokine
Cytokine Receptor
VEGF (Vascular
endothelial Growth Factor)
JAK (Janus-Associated
Kinase)
STAT (Signal Transducer
and Activator of
Transcription, or Signal
Transduction And
Transcription)
NF-B (Nuclear factor
kappa-light-chainenhancer of activated B
cells)
I-B (Nuclear factor of
kappa light polypeptide
gene enhancer in B cells
inhibitor)
Steroid
Steroid Receptor

Definition
Small cell-signaling protein molecules that are secreted by numerous cells
and are a category of signaling moleucles used extensively in intercellular
communication.
Receptors that are specific in binding to cytokines.
A signal protein produced by cells that stimulate vasculogenesis and
angiogenesis.
Family of intracellular, nonreceptor tyrosine kinases that transduce cytokinemediated signals via the JAK-STAT pathway.
Protein involved in regulation of growth, survival, and differentiation in cells.

Protein complex that controls the transcription of DNA, found in all animal cell
types and involved in cellular responses such as stress, cytokines, free
radicals, UV radiation, oxidized LDL, and bacterial or viral antigens.
Family of cellular proteins that inhibit the NF-B transcription factor.

Class of organic compounds that contain cycloalkane rings joined to each

other. They are small hydrophobic signaling molecules.
Found in the plasma membrane, cytoplasm, and nucleus, they are
intracellular receptors that can change gene expression over time period of
hours to days by initiating signal transduction when a steroid hormone binds
to the stated receptor.

28

Lecture Objectives
Take a brief look at a few other signal transduction systems that cells use to regulate gene
transcription.
Remember the TGF- signaling pathway, and that there are other pathways
that are involved. R-SMAD-SMAD4 complexes often cooperate with other
sequence specific transcription factors. The high affinity binding of SMADs
typically requires cooperation with other factors and can act with
coactivators or corepressors. From this, we know also that different target
genes are affected depending on the combination of SMAD and cooperating
transcription factor (cofactor). Thus, the regulatory input can occur at many
levels. There are factors that can
potentiate signaling of Type I and II
receptors, and ligands that can affect expression of cofactors. The
TGF- pathway is one of several that can affect transcription. There
are others.
There is cytokinase signaling through the JAK/STAT pathway.
Remember that cytokines are small proteins released by cells that
have specific effects on cells that have the appropriate cytokine
receptor. There are many, but cytokines include interleukins,
lymphokines, tumor necrosis factors, and the interferons. This is particularly helpful in immune response.
The JAK/STAT pathway has three components: (1) the cytokine receptor (with no intrinsic enzymatic
activity), (2) the Janus Kinase or JAK) (which associates with the receptor), and the Signal Transducer and
Activator of Transcription or STAT (which is the latent cytoplasmic transcription factor). STAT is not bound
to the receptor. There is a large family of specific cytokine receptors selectively expressed in different
types of cells. There are four types of JAKs, and seven types of STATs that are either homodimers or
heterodimers).
The pathway of the Cytokine JAK/STAT signaling pathway is executed in a certain manner. Ligand binding
to the receptor causes transphosphorylation (in the form of autophosphorylation), with STAT binding to
phosphotyrosine. Tyrosine phosphorylation allows the release of STATs. At the same
time, secondary STAT is phosphorylated via the growth factor receptor tyrosine kinase
pathway. The two pathways converge with the reciprocal binding of SH2 to the
phosphotyrosine, yielding a STAT dimer, called P-STAT. P-STAT dimer then enters the
nucleus, causing STAT activating the expression of various genes including SOCS1. The
expression product SOCS1 then exits the nucleus and then binds to the cytokine-JAK/STAT
complex and inducing a negative feedback mechanism, stopping the pathway. The
JAK/STAT pathway uses particular strategies, such as dimer formation, phosphorylation,
and subcellular localization. With cytokine signaling through the JAK/STAT pathway, an
activated JAK kinase phosphorylates a STAT, which then dimerizes and goes to the
nucleus. There can be crosstalk between the TGF- signaling and the cytokine JAK/STAT
signaling. Several signaling pathways can induce SMAD6 and/or SMAD7 expression,
including those in the JAK/STAT pathway.
Another pathway of particular interest is the NF-B pathway, in which signaling is triggered by bacterial cell
wall products (a form of innate immunity),
ProProToll-Like
inflammatory cytokines, viral infection, UV
Toll-Like
inflammatory
inflammatory
Activation
of
Receptors
Receptors
Activation of
cytokine
irradiation, B or T cell activation, which are
cytokine genes
genes
Bacterial
Cell
(Innate
the
Bacterial Cell
the NF-kappaB
NF-kappaB
(Innate
are
turned
on
are turned on
Wall
Products
Immunity
Signaling
Wall Products
Immunity
Signaling
by
NF-kappaB
forms of adaptive immunity. They are a family
by NF-kappaB
System
Receptors)
Receptors)
System
transcription
transcription
if five related transcription factors. In innate
factor
factor
immunity there is a process. In innate
immunity, involved in a certain manner. Signaling through the NF-B pathway involves NF-B being kept in
the cytoplasm until the signaling pathway is activated. I-B is the inhibitory subunit that binds to NF-B.
When the pathway is activated, I-B gets phosphorylated, which causes it to be targeted for destruction by
the proteosome. Now NF-B can go into the nucleus.

29

VEGF (vascular endothelial growth factor signaling) is

the third example of another pathway cells use to
regulate cell signaling. Mesodermal cells with VEGFR-2
utilize VEGF to develop into angioblasts, and then (with
more proteins and VEGF) to develop into the
endothelial cell tube. It utilizes a form of receptor
tyrosine kinase signaling. VEGF binds to the receptor
allowing the opening of extracellular domains. These
extracellular domains dimerize and kinase domains
phosphorylate each other. The receptor then binds and
activates PLC and then other effectors with SH2 or PTB
domains bind to the phosphotyrosines, and Ras and Raf
activation. These in turn catalyze MEK activation,
which then catalyzes ERK activation. This pathway of
activation is known as the MAP kinase pathway. From
there, the kinase from ERK activation moves to the
nucleus, allowing activation of the transcription factor and binding to the cells DNA causing the cellular
proliferation and differentiation. With
VEGF signaling, an activated kinase
goes into the nucleus and activates a
latent transcription factor by
phosphorylating it.
We can also utilize steroids as a
signaling tool. Steroids are small
hydrophobic (lipid-soluble) signaling
molecules. They can be found in the
cytoplasm or nucleus, and acts as the
receptor for the ligand and
transcription factor. This superfamily
of signaling molecules contains a DNA
binding domain, a ligand binding
domain (for direct binding) and
transcriptional activation domains. Steroids can pass through the plasma membrane and bind either to a
receptor in the cytoplasm or a receptor already in the nucleus (depending on the type of steroid). Steroid
binding allows the receptor to bind DNA and activate (or repress) the transcription of specific genes. They
are essentially free from the inhibitory state and can allow transcription. This can allow a contextdependent response to steroids. Certain receptors can coordinate the expression of several different
genes, depending on the presence of different cooperating transcription factors.
Compare and contrast these pathways with the TGF-.
Pathway
Strategies
Similarities
Differences
JAK/STAT
Heterodimer
Induced proximity model
The kinase in this system (JAK) is a separate
Formation,
applies
protein from the receptor (associated instead
Phosphorylation
of bound)
, and
Phosphorylation is the
Subcellular
key molecular switch in
Autophosphorylation (transphosphorylation)
Localization
this system.
mechanism with JAK rather than one receptor
type phosphorylating a different receptor
Invoves a change in the
type, as with TGF- receptors
localization of the
transcription factor from
Phosphorylation of the receptor is used to
the cytoplasm to the
recruit the binding of the transcription factor
nucleus.
to the receptor (which is not true of SMADs).
Dimer
There is a latent
Removal of an inhibiting subunit is required
NF-B
Dissociation,
transcription factor that
for activation instead of addition of a
Phosphorylation
accumulates in the
cooperating partner.
, and
nucleus when the
Subcellular
signaling pathway is
The inhibitory protein gets degraded in the
Localization
activated.
process.
VEGF
Phosphorylation
Induced proximity model
Autophosphorylation (transphosphorylation)
Signaling
applies.
mechanism with VEGFR rather than one
receptor type phosphorylating a different
Phosphorylation is a key
receptor type as with TGF- receptors.
molecular switch.
Involves a change in the localization of the

30

Steroid
Signaling

Ligand Binding,
Subcellular
Localization

Change in the
transcription factor
localization from
cytoplasm to nucleus (in
some cases).
Steroid receptors also
participate in
cooperative interactions,
like SMADs.

kinase from the cytoplasm to the nucleus

(where it phosphorylates gene-specific
transcription factors).
Ligand passes directly through the membrane
instead of binding to a receptor in the
membrane.
The steroid receptor is itself the gene-specific
transcription factor.
Ligand binding directly affects the
transcription factors and is the primary switch
mechanism, not phosphorylation. (Some
receptors are further regulated by
phosphorylation.)

Understand the variety of ways that cells regulate transcription factors.

From these pathways, we can note that there is a common theme of latency of the transcription factors. In
order for these signaling pathways to start having an effect, the cell need only to activate a latent
transcription factor that is already present and ready to go. If the cell had to first make the transcription
factor from scratch, the responses would not start as quickly and this could be deleterious and inefficient.
Giving the only function of activating the latent transcription factor is such a boon that the cell can
regulate these several transcription factors simultaneously and in response to intracellular to extracellular
demands.

Lecture XXVIII
E xperimen ta l Too l: Tra nsg en ic Appr oa ches in Physiolog y

Terminology
Term
Genotype
Phenotype
Wild-Type
Mutant
Constitutively Active
Transgenic Mouse
Transgene
Random Insertion
Founder Mouse
Chromosomal DNA
Promoter
Constitutive Promoter
Inducible Promoter
Coding Sequence
Protein of Interest
Reporter Gene
Pronucleus
Zygote
Surrogate Mother

Definition
Genetic makeup of a cell, specific to a character in consideration.
Observable characteristic or trait
Phenotype of the typical form of an organism occurring in nature.
Individual, organism, or genetic character arising from mutation.
Protein whose activity is constant and active, one that transcribed from the
DNA all the time.
Mouse that has had its genome altered by genetic engineering.
Gene or genetic material that has been transferred naturally or by genetic
engineering. Describes a segment of DNA containing a gene sequence
isolated from one organism and introduced into a different organism.
Insertion of DNA segment into an unpredictable portion of the chromosome.
Laboratory mice that have been produced from a genetically manipulated
egg or embryo.
DNA compacted into a chromosome. In prokaryotes, it is compacted into a
circular form known as the plasmid.
Regulatory region of DNA usually located upstream of a gene, providing a
control point for regulated gene transcription.
An unregulated promoter that allows for continual transcription of its
associated gene.
Type promoter in which their activity is induced by the presence or absence
of biotic or abiotic factors.
Portion of a genes DNA or RNA, composed of exons that codes for protein.
Protein under study by a researcher.
Gene that researchers attach to a regulatory sequence of another gene of
interest in bacteria, cell culture, animals or plants.
Nucleus of a sperm or egg cell during the process of fertilization, after the
sperm enters the ovum, but before they fuse.
Initial cell formed when two gamete cells are joined by means of sexual
reproduction.
Mother that is carrying the embryo of that does not have the mothers DNA, a
foreign embryo.

31

Polymerase Chain
Reaction (PCR)
Agarose Gel
Electrophoresis

Scientific technique to amplify a single or a few copies of DNA across several

orders of magnitude, generating thousands to millions of copies of a
particular DNA sequence.
Method used to separate proteins by charge or size in a medium consisting of
agarose.

Lecture Objectives
Learn about transgenic and knockout mouse approaches in physiological studies.
Unfortunately, because the use of human subjects in experiments is ethically questionable though the
motives are typically altruistic, scientists have to model human diseases in animal subjects. It can be done
by studying the physiology of a normal animal or tissue and then altering the system in some way to gain
insight into the normal physiology. We can do this in several ways: (1) by exposing the animal to different
Suppose'you'think'that'protein'B'fits'into'a'
physiological situations, (2) treating the animal with agents that affect protein
function (inhibitors or
activators) or agents that affect gene expression, or (3) altering the genetic
makeup (genotype) of an
physiological'pathway'as'follows:'
animal. We can do this with two different types of mouse subjects: transgenic and knockout mice.

One*way*to*test*the*hypothesis.*
For example, suppose there is a pathway, and a scientist is
A'
B'
C'
D'
investigating the pathway that signals in a sequential order, with the
signal causing expression of A, then B, the C, etc. The scientist can
Signal'
run two experiments, either by manipulating the genetic makeup so
that one of the coding proteins is inactivation (causing downstream
A'
B'
C'
D'
inactivation) or by increasing the gene expression of one of the
Hypothesis:''Signal'acFvates'A'which'acFvates'B'etc.'''
proteins, so that the upstream sequence would be inactivated. To
Signal'
explain the downstream inactivation, the genetic makeup is altered
so that B is no longer present, and (predictable) C and D should not be
Another*way*
1:''If'we'alter'the'geneFc'
A' to*test*
B' the*
C' hypothesis
D' active.*
even when the signal is present,Experimental'test'#
even when A is still
activated by
makeup'of'the'animal'such'that'
the signal. The upstream inactivation can
be done by altering the B'is'no'longer'
Signal'
genetic makeup of the animal so the B present,'
is expressed
a form that is
C'and'in
D'should'not'be'acFve'even'when'
C and D remain
active, but
A'
B'
C'
D' active all the time (constitutively) and then
the'signal'is'present.''
A'should'sFll'be'acFvated'by'
To'perform'this'test,'we'need'to'be'able'to'
not A, even with the absence of the signal. These two strategies work,
*'
eliminate'the'funcFon'of'
B
.''One'way'is'to'make'a'
the'signal.''
B'
Signal'
but require the introduction of the altered product into the animal.
knockout'animal'in'which'the'gene'encoding'
Introduction
of a foreign gene into theB'animal is necessary to make a transgenic animal. The transgenic
has'been'inacFvated.''More'on'this'in'the'next'
mouse has altered
gene expression into the animal, and the knockout mouse has elimination of function in
Experimental'test'#
2:''If'we'alter'the'geneFc'makeup'
lecture.'
one
of the proteins.
of'the'animal'such'that'
B'is'expressed'in'a'form'that'
is'acFve'all'the'Fme'(consFtuFvely'acFve),'then'C'and'
It is also important to remember what are a genotype, a phenotype, and the difference between wild-type
D,'but'not'A'should'also'be'acFve'all'the'Fme'(even'
and mutant. Genotype is the genetic constitution of an individual cell or organsm and the associated
in'the'absence'of'the'signal).''

alleles at one or more specific loci. The phenotype is the observable characteristics of a cell or organism.
A wild-type organism is the normal and common form other organism while the mutant is a changed or
abnormal form.

Making*a*transgenic*mouse*

Know about the basics about how transgenic mice are produced.
Microinject#
DNA#
Transgenic and knockout mice are utilized as research
into#
ferBlized#
tools. To understand a gene of interest, the gene is
eggPlasmid'DNA'
#
Fo,#
Founder#
transgenic#
mouse#
injected into transgenic or knockout mice with the
Embryos#
genotype manipulated. From there, a phenotype is
generated, and thus the function of a gene is learned.
Transfer#
InserBon*
Offspring# of*transgene*into*a*
A transgenic mouse is a mouse with foreign DNA known
embryos#
Chromosome*
in*the*ferBlized*egg*
as a transgene (engineered by the experimenter)
Cooper,'Figure'3.37'
Transgene'DNA'
inserted into the chromosomal DNA. It is typically occurring by random insertion
inserts'into'the'
into a chromosome of the fertilized egg. Making a transgenic mouse is
chromosomal'
beneficial because it allows the experimenter to express an altered gene product
DNA,'but'not'at'
any'parFcular'site.''
or a normal one that is expressed at the wrong time, place, or amount. It can
It'goes'in'
Transgene#
allow interpretation on function, phenotype of the transgene expression as well
randomly.''
DNA#
the physiological pathways affected in the subject, and what parts of the protein
InserFon'does'not'
require'matching'
important in the function. These interpretations can allow application into
up'of'sequences.'
disease models. This can not only be done in mice, but also bacteria, flies, fish,
Chromosomal#
plants, yeast, birds, cows, worms, frogs, and pigs.
'DNA#
You can get a transgene into every cell of a mouse by microinjection of the transgene DNA into the
pronucleus of a mouse zygote. After fertilization, there is a brief period before the nucleus fuses. The
predecessor to this newly developed nucleus is the pronucleus. The injected mouse zygote from there is
implanted into a surrogate mother that carries the organism to term. This has a fairly good success rate
with stable integration of transgene observed in 10-40% of mice and each founder mouse can be used to
establish an independent line of transgenic mice.

32

How*can*we*control*when*and*where*the*
transgene*is*expressed*in*the*animal?*
Transgene'

Know the basics about the composition of transgenes.

Promoter'
Gene'X'
Plasmid'
A transgenic mouse has foreign DNA (transgene) present in the chromosomal DNA
Cut'out'
linear'
of every cell. The DNA is injected and inserted randomly. Transgene DNA inserts
fragment'
Inject'DNA'
into a chromosomal DNA, but not at any particular site. It goes in randomly.
into'zygote'
Insertion does not require matching up of sequences. Once in a chromosome, a
transgene is usually passed on to successive generations. The question is now how the experimental can
control when and where the transgene is expressed in the animal. An experimenter and cut out a
PCR*method*from
for*detecBng*
* and then place it into the transgene with promoters and inject it into the zygote.
fragment
thetransgene*
plasma
The promoter is the sequence in the DNA to which RNA polymerase binds to
Primer'
Mouse'tail'DNA'
Tg'
begin transcription. The promoter is used to influence where and when the
Primer'
transgene is expressed and at what level of expression. The promoter can be
Polymerase'Chain'ReacFon'
PCR'
a well-characterized promoter region from a gene that directs a specific
pattern of gene expression, a constitutive promoter, an inducible promoter, a
gene promoter region under study. The gene
Run'on'an'agarose'gel'
can be a sequence encoding the protein of
interest, a sequence encoding a reporter gene, Cre recombinase, or other
possibilities.
'

'

Understand how the PCR method can be used to detect the presence
of transgenes.
This falls into the question of how to determine which mice exhibit the
transgene. The general idea of the PCR method is to amplify the region of the
transgene with boundaries known as primers. From there, an experimenter
can run it on an agarose gel and observe the presence of the isolated copies
of DNA segment.

Lecture XXIX
E xperimen ta l Too l: Kno ckou t M ic e (Tar geted G ene Ina ctiva tio n)

Terminology
Term
Knockout Mouse
Forward Genetics

Reverse Genetics

Allele
Embryonic Stem Cells
Pluripotent
Homologous
Recombination
Gene Targeting Vector
Region of Homology
Drug Resistance Gene
Selection Strategy
Screening Strategy
Positive and Negative
Selection

Definition
A mouse whose DNA has been genetically engineered so that it does not
express particular proteins
Approach that encompasses several means of identifying the gene or set of
genes that are responsible for a particular phenotype within an organism .
Forward genetics can be thought of as a counter to reverse genetics, which
seeks to alter genes in order to illuminate their multiple phenotypes
An approach to discovering the function of a gene by analyzing the
phenotypic effects of specific gene sequences obtained by DNA sequencing.
This investigative process proceeds in the opposite direction of socalled forward genetic screens of classical genetics. Simply put, while forward
genetics seeks to find the genetic basis of a phenotype or trait, reverse
genetics seeks to find what phenotypes arise as a result of particular genes.
One or two more forms of a gene or a genetic locus.
Pluripotent stem cells derived from the inner cell mass of the blastocyst, an
early-stage embryo. Distinguished by their pluripotency and their ability to
replicate indefinitely.
Refers to the ability to differentiate into any of the three germ layers:
endoderm, mesoderm, and ectoderm.
Type of genetic recombination in which nucleotide sequences are exchanged
between two similar or identical molecules of DNA.
A bacteriophage, plasmid, or other agent that transfers genetic material from
one cell to another.
Traits that are common among organisms due to sharing a common ancestor,
and such traits have similar embryological origins and development.
Genes in an microorganism which confer resistance to antibiotics, through
manipulation of surface proteins or the antibiotics target.
Methods to enrich for correctly targeted cells.
Diagnostic tests to identify correctly targeted cells.
Positive Selection: Select for ES cell that have taken up a drug resistance
gene included in the targeting vector.

33

Neo Gene (Neomycin

Resistance Gene)
TK Gene (Thymidine
Kinase Gene)
Restriction Enzyme Digest
Southern Blot
Agarose Gel
Electrophoresis
Blastocyst
Surrogate Mother
Chimera
Germline Transmission
Conventional Gene
Targeting
Conditional Gene
Targeting
LoxP (Locus of Crossover
in P1)
Floxed Gene
Cre Recombinase
Tissue Specific
Recombination

Negative Selection: Select against ES cells that have randomly integrated the
targeting vector.
Gene that codes for neomycin resistance in cells.
Gene that codes for the phosphotransferase (kinase) that is found in most
living cell, which catalyze the phosphorylation of deoxythymidine to
deoxytymidine 5-phosphate.
Use of restriction enzymesto cut or digest DNA at specific recognition
nucleotide sequences known as restriction sites.
Method utilized for detection of a specific DNA sequence in DNA samples. It
combines transfer of electrophoresis-separated DNA fragments to a filter
membrane and subsequent fragment detection by probe hybridization.
Method used to separate proteins by charge or size in a medium consisting of
agarose.
Strcture formed in the early embryogenesis of mammals, after the formation
of the morula.
Mother that is carrying the embryo of that does not have the mothers DNA, a
foreign embryo.
Single organism that is composed of two or more different populations of
genetically distinct cells that originated from different zygotes involved in
sexual reproduction.
Transmission of the targeted gene copy to the offspring if the embryonic stem
cells contributes to the germline (gonads).
Gene is inactivated in all cells at all times.
Gene ablation is cell type-specific or inducible.
Specific sites in a DNA molecule for Cre protein that surround a directional
core sequence where recombination can occur.
Sandwiching of a DNA sequence between two lox P sites and is a contraction
of the phrase flanked by LoxP.
Tyrosine recombinase enzyme derived from the P1 Bacteriophage that carries
out site-specific recombination events.
Expression of certain genes in specific tissue, with effects only occurring in
cells expressing certain genes.

Lecture Objectives
Know why knockout mice are useful in physiological studies.
Knockout mice have an endogenous gene of interest that is inactivated (knocked out). In variations of this
technique, a gene can be modified or replaced with a different gene. Why do researchers make knockout
mice? It allows the determination: (1) which physiological functions are lost when the specific gene is
altered, (2) which functions are not affected, (3) the kinds of adaptive or maladaptive changes result when
the gene is altered, (4) whether the phenotype mimics a disease of
interest. In a bigger picture, knockout mice utilize an element of
reverse genetics, in which there is a gene attained and the
researcher wants to determine what are the effects of a mutation
(genotype to phenotype), as opposed to forward genetics, in which
there is a mutant phenotype to determine which gene was mutated
(phenotype to genotype). It allows the determination of whether it
fits the hypothesis or not.
Learn how knockout mice are generated.
Knockout mice are generated from embryonic stem cells in culture.
Embryonic stem cells are pluripotent stem cells that can be
maintained in an undifferentiated cell culture. An altered version of
the target gene constructed by genetic engineering is introduced
into each embryonic stem cell, and them each cell proliferates to
form a colony. The colony is then tested for whether each cell has
had the DNA fragment replace one copy of the normal gene. Then we need to prepare the female mouse
for uptake. The female mouse mates, waits 3 days and harvest early embryos. The embryonic stem cells
are then injected into the early embryo. The hybrid early embryo is partially formed from embryonic stem
cells. The hybrid is then introduced into the pseudopregnant mouse until birth. The somatic cells of the
offspring are then tested for presence of the altered gene and then selected mice are bred to test for gene
in germ-line cells. This process can yield a transgenic mouse with one copy of the target gene replaced by
altered-gene in germ line.

34

Know about homologous recombination.

Gene targeting (knockout) technology depends on pairing up and
recombination between homologous sequences of DNA, known as
homologous recombination. The recombination involves
switching (a crossing over) of a chromosome between certain
gene targeting vectors. Basically, there is a crossing over into
the targeting vector and then a return to the homologous
sequence. The crossing must be done twice or face the
consequence of incomplete crossover.
A major challenge of gene targeting is that mammalian cells
undergo a high frequency of random integration events in
addition to the more rare targeted events. If random insertion
occurs, then the gene of interest is not knocked out. Random
insertion occurs more frequently than homologous
recombination.
Understand the strategy of positive/negative selection.

ES
ES Cells
Cells

Introduce
Introduce targeting
targeting
vector
vector by
by
electroporation
electroporation

Select
Select for
for colonies
colonies
that
that survive
survive both
both
positive
and
positive and
negative
selection
negative selection
drugs
drugs

Expand
Expand those
those rare
rare
surviving
surviving colonies
colonies
into
into separate
separate cell
cell
lines
lines

Screen
the candidate
candidate
Screen the
cell
lines for
for those
those
cell lines
that
really
do
that really do have
have
the
the gene
gene ofof interest
interest
correctly
correctly targeted
targeted

This leads to the question how scientists can separate the correctly targeted embryonic stem cells from
the ones that result from random integration. We can utilize selection strategies (ways to enrich for
correctly targeted cells) or screening strategies (diagnostic tests to identify correctly targeted cells. One
selection involves positive and negative selection. Positive selection selects for embryonic stem cells that
have taken up a gene included in the targeting vector. Negative selection selects against embryonic stem
cells that have randomly integrated the targeting vector.
Learn about Southern blotting as a method to test for
correct gene targeting.
Restriction enzyme digestion and Southern blotting can be utilized to
check if the gene of interest has been correctly targeted. Southern
Blotting simply looks at the structure of DNA at the gene of interest
before and after correct targeting has occurred. It can be used to
analyze the DNA of embryonic stem cells ot check if the correct gene
has been targeted. The DNA from individual embryonic stem cell
clones is digested with an enzyme. The digest samples are then run
by agarose gel electrophoresis to separate these digested proteins.
From there, a researcher can determine which cells have a correctly
targeted embryonic stem cell clone.
The overall summary is that through a combination of restriction enzyme digestion,
transfer to nitrocellulose paper, and DNA probe hybridization, one can check for the
expected structural changes in the gene of interest. Basically, this is to confirm that
the changes in DNA that were expected to occur (when gene targeting is done
correctly) actually happen?
If a scientist did get embryonic stem cells that are correctly targeted, what will he or
she do with it? From there, the embryonic stem cells that have been correctly targeted
are then injected into a normal mouse embryo (at the blastocyst stage). Several
embryos are then placed into a surrogate mother. The embryonic stem cells mix
together with the normal cells and then contribute to various tissues, producing
chimeras. Chimeras are altered foreign cells that are incorporated into the normal
embryo. If the embryonic stem cells contribute to the germline (gonads) of the
chimeric mouse, then the targeted gene copy might be transmitted to the offspring.

35

How!to!get!homozygous!knockout!mice!

From there, we can get two types of knockout mice, either

heterozygous. Heterozygous mice will have only one of the
knocked out, while homozygous will have both copies

+/T!

+/T!

homozygous or
Digests&
copiesKpnI&
that
are
knocked out.

This is a form of conventional gene targeting. However,

there is a small
problem with conventional gene targeting. It may not be
that useful. The
gene of interest can be expressed in multiple cell types, and
will not allow the
Southern!blot!of!
determination of whether the phenotype is due to ablation of
the mouse!tail!DNA!
gene function in
T/T!
+/+!
+/T!
!
one cell type versus the other. Eliminating the gene
everywhere could be
Interbreed!mice!carrying!Floxed!gene!with!transgenic!
mice!expressing!Cre!recombinase!in!specific!;
lethal to the embryo during development, and it is irreversible. It does
not allow the deletion of the ssue!
gene
in the adult form, when the tissue is already fully formed.
Know the basics about conditional gene targeting
sing the cre-IoxP system.
Conditional gene targeting involves gene ablation that is
cell type-specific or inducible. One can utilize conditional
targeting in the Cre/LoxP system. LoxP is in the
chromosome, and is involved in the removal of the gene
segment from the chromosome. Cre recombinase is
expressed only in specific tissue. The recombination occurs
in a tissue specific manner. The new mouse strain should
have the floxed gene and the Cre transgene. The LoxP sites
recombine only in cells expressing Cre recombinase.

Recombina; on!occurs!in!a!; ssueT

Cre&
transgene&
floxed&
gene&
specific!manner!
New&
mouse&
strain&
Note:!loxP!sites!in!this!
Interbreed!
contains&
floxed&
gene&
example!are!placed!in!
lines! and&
Cre&
transgene&

introns!flanking!a!cri; cal!
exon.!

Lodish,!Fig!8T35!

loxP&

loxP&

The Cre-LoxP system is a three-step process that consists

loxP!sites!recombine!
of:
only!in!cells!
1. Making mice in which the gene of interested is floxed with the recombination sites.
Using
expressing!Cre!
convention gene targeting methods does this.
recombinase!
Lodish,!Fig!8T35!
2. Make a different mouse line (transgenic mice) that expresses the Cre recombinase enzyme in
specific target tissue.
3. Interbreed mice carrying Floxed gene with transgenic mice expressing Cre recombinase. This
yields mice that have the floxed gene (meaning that it has LoxP) and transgene.

Lecture XXX
T GF- S ign alin g Kn oc ko ut M ic e, Dis eas e Mod els, an d Ca se Con clusion

Terminology
Term
Hyperdilation
Balance Model
Knock in versus Knockout

Reporter Gene
LacZ or -gal Reporter
Gene
Luciferase Reporter Gene
Endothelial-Specific
Knockout
Homozygous Knockout
Heterozygous Knockout
BMP (Bone Morphogenic
Protein)
Modifier Genes

Definition
Excessive dilation of the blood vessels.
Regulation in the transition from one phase to another of a process. Assumed
that ALK1 and ALK5 were both expressed in endothelial cells.
Knock-in: Genetic engineering method that involves the insertion of a
protein coding cDNA sequence at a particular locus in an organisms
chromosome.
Knockout: Genetic engineering of an existing gene by replacement or
disruption with an artificial piece of DNA. Knock-in is similar to knockout, but
replaces a gene with another instead of deletes it.
Gene that researchers attach to a regulatory sequence of another gene of
interest in bacteria, cell culture, animals or plants.
Reporter gene that encodes -galactosidase, which cause bacteria that
express the gene to appear blue in a media containing X-gal.
Gene utilized as a laboratory reagent that yields a class of oxidative enzymes
utilized in bioluminescence and distinct from a photoprotein.
Strategy that involves making mice that lack ALK1, ALK5 or TGFTII gene and
to observe effects in vascular endothelial cells.
Genotype where both alleles have the same inactivated gene.
Genotype where one of the alleles have the inactivated gene.
Group of growth factors that orchestrate tissue architecture throughout the
body.
Segment of DNA that is involved in producing a polypeptide chain. It can
include regions preceding and following the coding DNA as well as introns

36

between the exons.

Lecture Objectives
Know which genes are mutated in HHT and how these genes fit into the TGF- pathway.
Remember that hereditary hemorrhagic telangiectasia was covered from a
systemic to a molecular level, and the mutations that cause HHT are
attributed to the TGF- signaling pathway, and affecting gene targeting..
There is most likely a mutation either in the ALK1 receptor or the CoSMAD. There are several types of HHT, but HHT type 2 is the one involved
in the mutations in the Activin receptor-like kinase 1 (ALK-1) gene, which
is a Type 1 TGF- superfamily receptor. How does the defect in the
signaling pathway result in specific vascular defects in hereditary
hemorrhagic telangiectasia? We know that the genetic defects most likely
result in aberrant endothelial cell responses to specific signals, including
dysregulation of a variety of genes in endothelial cells. We can know where the problem is via a
microarray. We can isolate the mRNAs from normal and HHT cells and then compare with probes.

The current ideas about HHT involve dysregulation of the specific genes
that could result in abnormal production of the extracellular matrix,
altering cell adhesion and migration in angiogenesis. This can result in
irregular vessel formation, impaired recruitment and ifferentiation of
mesenchymal cells into smooth muscle cells.
Understand how knockout mice and transgenic mice can help us
to understand and mimic disease.
Knockout mice were utilized in with two methods: conventional/global
knockout and conditional/tissue-specific knockout. Conventional gene
targeting was utilized to make a global knockout. The conventional
(homozygous) knockout of ALK-1 resulted in the vascular abnormalities
with embryo death at mid-gestation. The ALK-1 Knockout spurred
vascular abnormalities. It yielded the following findings: (1) excessive
fusion of capillary plexes into cavernous vessels, (2) hyperdilation of
large vessels, (3) deficient differentiation and recruitment of vascular
smooth muscle cells, and (4) enhanced expression of angiogenic factors
and proteases.
Initially, HHT was attributed to
homozygous phenotypes for HHT, but scientists made a more thorough
investigation to show that mice that were heterozygous for the mutation
in ALK-1 exhibit age-dependent vascular lesions similar to those in
humans. Transgenic methods were used to test and counter the
assumption of the Balance Model in mice.
Mice were also utilized as a media for a tissue-specific knockout strategy that was discussed in the
previous strategy. This utilized an endothelial cell-speciic knockout strategy, in which mice are made or
obtained with a floxed verison of ALK1, ALK5, or TGFRII genes, all in separate experiments. Scientists
would
make a transgenic mouse that would
express Cre recombinase only in vascular endothelial cells,
Hypothesis:!!Balance!model!for!TGF9
!
and interbreed the transgenic Cre expressing mice with each of the floxed mouse lines, and obtained the
signaling!in!the!regulaEon!of!angiogenesis!
mice with floxed gene as well as the Cre transgene.
In!endothelial!cells,!
Learn about the evolution of ideas describing TGF- pathway signaling
ALK1!might!normally!!
regulate!the!transiEon!
during vascular maturation and in HHT.
from!the!acEvaEon!phase!
The first model that was presented was the Balance Model. Initially it was thought
to!the!resoluEon!phase!of!
that endothelial cells in blood vessels express both ALK1 and ALK5, essentially two
angiogenesis.!
different type I receptors. In endothelial cells, ALK1 might normally regulate the

ResoluEon!

transition
from the activation phase to the resolution phase of angiogenesis. In

In!ALK1!KO!mice,!the!
ALK1 knockout mice, the defects would result from the abnormal persistence of
defects!would!result!from!
the activation phase. Essentially, based on that model, HHT was from too much
the!abnormal!persistence!
activation and not enough resolution. The balance model assumed that ALK1 and
of!the!acEvaEon!phase.!

AcEvaEon! ALK5 were both expressed in endothelial cells.

S.!P.!Oh,!et#
al.,#
PNASs#
97,$2626$(2000).!

37

To test the expression pattern of ALK5

approach, one copy of the ALK5 gene
the lacZ gene was inserted into that
reporter. The same experiment was
ALK1. Experimental performance
stated that ALK1 was expressed in
cells, but ALK5 is actually expressed in
muscle cells, countering the assumption
Model.

using a reporter gene

was knocked out and
ALK5 locus as a
done to replace the
yielded results that
vascular endothelial
vascular smooth
made by the Balance

With the newly revised model of TGF- signaling during vascular maturation, we know that ALK1 was
expressed in the endothelial cell while ALK5 receptors are in the smooth muscle. Scientists can predict
that conditionally knocking out ALK1, ALK5, and TGFRII in endothelial cells would yield the following
results:
Knockout of ALK1 results in severe malformations mimicking the pathologic features of HHT
(expected).
Knockout of ALK5 resulted in not exhibiting vascular defects (expected).
The!new!model!incorporates!BMP9/BMP10!and!their!
Type!II!receptors!that!can!interact!with!ALK1!in!vascular!
Knockout of TGFRII did not show these vascular defects (which was not expected).
endothelial!cells!

The conclusion generated from this is that ALK1, not ALK5 and TGFRII, is
necessary in endothelial cells for the signaling that is pertinent to the
pathogenesis of HHT. Since the TGFRII knockout did not mimic HHT, this
suggests that the ligand for ALK1 signaling might not actually be a member of
the TGF- subfamily. Other experiments with endothelial cells in culture
indicated that BMP9 and BMP10 were actually better candidates to be the
ligand for ALK1 than TGF-1.
So far, the most recent
model shows incorporation of BMP9 and BMP 10 and
their Type II receptors that can interact with ALK1 in
vascular endothelial cells.
Realize that we do not yet understand
everything about HHT.
There are still, unfortunately, lots of questions about
HHT. Why are the lesions in HHT so localized in the
affected person? Is a second hit event necessary to
initiate a local lesion? Could malformations be related
to abnormal responses to local injury? We also dont know why is the severity of the disease so variable
between affected individuals? Are there modifier genes that can influence how severely the disease
presents itself? Why are the key target genes that are abnormally expressed in HHT? Do nontranscriptional effecs also contribute? How do defects like arteriovenous malformations actually form?
To address the plethora of questions and plausible hypotheses, HHT researchers have made some
inducible knockouts, when the gene is only knockout when an inducer is given. One group made an
inducible knockout of ALK1 so they could get adult mice and then induce ALK1 knockout. They found that
wounding can induce the new formation of arteriovenous malformation in the ALK-1 deleted mice. The
dorsal skinfold chamber model can be used to follow the same blood vessels over time. Using the dorsal
skinfold window chamber system the development of an arteriovenous malformation in response to
wounding was followed over time. This may help to understand how the lesions develop.
So whats the point? There are some good mouse models of HHT and these can be used to test ideas
about the factors that influence the formation of vascular defects. From what we understand so far, we
also have potential therapeutic targets. There has been success with drugs that inhibit the VEGF signaling
pathway, with VEGF elevation is present in the skin telagiectatic lesions of HHT patients. Scientists have
remembered that notch signaling along with VEGF-A signaling, is one of the key regulators of tip cell
formation during angiogenesis. Studies have also shown that Notch4 normalization reduces the blood
vessel size in arteriovenous malformations. Thus, in conclusion, there is still much to be learned and
researched about hereditary hemorrhagic telangiectasia. Studies such as those previously stated
unraveled more possible causes for hereditary hemorrhagic telangiectasia.

38