Developmental Biology: Final Exam Study Guide

11/18

1. Somites generate several differentiated cell types:
a. Cartilage
b. Muscle
c. Bone
2. Somites develop from the somitocoel- a single cell thick, hollow portion
3. The initial somite that forms is called the epithelial somite.
a. The epithelial somite develops into the mature somite.
b. The epithelial somite is: a hollow ball of cells
c. The mature somite: has different layers and makes a dermomyotome
and a sclerotome
d. Dermomyotome generates: dermis and muscles which are tightly
packed structures.
e. Sclerotome: bone and cartilage which are loosely packed
mesenchymal structures.
4. What is one of the sources of bone and cartilage in your body from somites?
Sclereotome
5. What are the 3 different sources of bone in your body?
a. (Somitic) Sclerotome: Makes axial skeleton
b. Lateral plate mesoderm: Makes bones in limbs
c. Neural crest cells: Make bones in head
6. What is osteogenesis?
a. Development of bone
7. What are the 2 major ways to develop bone (osteogenesis)?
a. Endochondrial osteogenesis: Forms from a cartilage model of
ossification through a cartilage intermediate. You first make cartilage
and then you make bone through ossification of cartilage.
i. This is the way that most of the bones in your body is made.
Most bones in your body come from a cartilage matrix.
ii. Bone growth: long bones in limbs and most of axial skeleton
forms through endocondrial ossification
iii. What is the extra cellular matrix of bone like versus cartilage?
Bone has a hard calcium ECM while cartilage has a flexible
ECM.
iv. Make chondrocytes first then generate osteoblasts
b. Intramembranous osteogenesis: direct deposition of bone matrix via
osteoblasts
i. Craniofacial skeleton forms intramembranous ossification.
8. Cell types
a. Osteoblasts: secrete bone matrix (ECM)
b. Chondrocytes: secretes cartilage matrix (ECM)
c. Pericondrium: surround cartilage. Some differentiate into osteoblasts.
Make important signaling molecules needed for bone and cartilage.
9. Overview of Endochondral ossification: Making, generating, lengthening
cartilage. Chondrocyte specificationPre-cartilage
condensationGrowth/ProliferationDifferentiation into osteoblasts
a. Where are cartilage precursor cells derived from? Somitic sclerotome,
lateral late mesoderm, or neural crest cells
b. Chondrocytes must first be specified. After specification, a cartilage
condensation is formed.
c. What happens during pre cartilage condensation? Loosely aggregated
mesenchyme (often migratory) skeletal precursors cells must pack
tightly into a new form that models the location and shape of the
skeletal elements. The specificed chondrocytes are cartilage
progenitors that make the compact clusters, called pre cartilage
condensations and that dictate the location of the future bone.
d. Bony growth occurs after Chondrocyte specification. It is driven by
proliferation. The function of proliferation to drive the lengthening of
cartilage and the increase in size (cells turn into prehypertrophic
chondrocytes).
e. After growth/proliferation, the pre hypertrophic chondrocytes will
either generate osteoblasts or undergo apoptosis.
10. Specification of Chondrocytes
a. Transcription factor Sox9 is necessary for specification of
chondrocytes.
b. Sox9-/- Loss of cartilage
c. In humans, mutations in Sox9 have Campomelic Dysplasia with short
limbs and sexual defects (autosomal sex reversal).
d. What does Sox9 do? Sox9 directly activates transcription of Collagen
type II, a ECM protein necessary for cartilage formation.
11. Generation of Proliferation chondrocytes that cause growth of
element/Differentiation of osteoblasts
a. How is endocondral bone lengthened? Endochondral bone is
lengthened by adding cartilage and then ossifying that cartilage.
b. Lengthening of cartilage comes from proliferative chondrocytes.
i. Indian Hedgehog (IHH) and Parathyroid hormone related
protein (Pthrp) protein.
ii. Lengthening is due to the maintenance of cartilage. Even in
adolescence you have the cartilage growth plate which is
where bone growth occurs.
iii. Distal tip of bony element has slowly proliferating
chondrocytes and the proximal tip has the most highly
proliferating chondrocytes. Where does more proliferation
occur? Proximal tip of bony element. The Proximal tip also has
columns of highly proliferating chondrocytes. Individual cells
have columnar appearance.
iv. What is driving the growth of the bone? Highly proliferative
region
v. As the cells proliferate and come out of the high proliferative
columnar region, they begin to undergo hypertrophy.
Hypertrophy is when the cells become larger.
c. Pre-Hypertrophic cells are greatly enlarged and have one of two fates.
The Prehypertrophic chondrocytes will: a)under go apoptosis,
programmed cell death or b) differentiate into osteoblasts and
become secreting bone matrix.
12. What is driving proliferation?
a. Ihh-/- : condensation has happened normally, but the bone doesnt
lengthen. 2 defects:
i. The bones are shortened because you have a massive decrease
in the proliferation of chondrocytes.
ii. Loss of PTHrp Undergo precocious ossification because of a
loss of Pthrp in Ihh mutants.
b. Pthrp mutants: precocious ossification and shortening of skeletal
elements. In humans, the pthrp mutant causes a specific type of
dwarfism.
13. Feedback loop between Pthrp and Ihh are responsible for growth of te bone.
i. Pthrp is expressed in the slower proliferating chondrocytes
and the hypertrophic chondrocytes are expressing Ihh. Ihh in
the hyperrophic chondrocytes signals to the proliferative cells
to maintain proliferation in the prehypertrophic chondrocytes.
Ihh also maintains the expression of Pthrp. Pthrp blocks the
transition into a hypertrophic chondrocyte. Pthrp maintains
cells in the proliferative state. Cells that move out of the Pthrp
region allow them to turn into hypertrophic cells. Prevent
them from undergoing hypertrophy maintains a region of
proliferation that drives bone growth. Ihh promotes growth by
preventing differentiation.
14. Differentiation of osteoblasts: Proliferation to hypertrophy
a. What is responsible for the transition from proliferation to
hypertrophy? Chondrocytes that have been specified and are
undergoing proliferation express transcription factor Sox9 for
specification and Ihh and Pthrp for proliferation. Whenever these cells
stop dividing and start differentiating, they are called pre-
hypertrophic cells.
b. What do prehypertrophic and hypertrophic cells express? HDAC4
(Histone deacetylase 4) and Runx2 T.F. as they mature. HDAC4 --|
Runx2. Expressed more strongly in prehypertrophic cells.
c. What is the function of a histone deacetylase? The histone deacteylase
regulates gene expression.
d. Runx2 is T.F. that is expressed in the prehypertrophic but more
strongly in the hypertrophic chondrocyte cells.
e. HDAC4-/- Precocious ossification and elevation of Runx2.
f. HDAC4+/+ --| inhibits Runx2 in the prehypertrophic region since
HDAC4 is strongly expressed here. As HDAC4 levels are weak, Runx2
levels are elevated in the hypertrophic region.
g. Runx2 -/- No formation of bone and failure to ossify. Limbs are
shortened because Runx2 is required to maintain Ihh expression. Loss
of Osx and Ihh.
h. Runx2 +/+ is necessary for the expression of Osx (T.F.) expressed by
differentiated osteoblasts.
i. Osx2 -/- No bone development but shortening of limbs does not
occur.
j. The function of Osx is to cause the cells to secrete Collagen type I
which is a bone specific ECM and generated mineralized Ca2+ based
bone.




11/20

1. Bone Homeostasis
a. What is bone homeostasis? Maintaining the proper amount of bony
material.
b. What is bone homeostasis maintained by? The relative activity of
osteoblasts that make bone and osteoclasts that destroy bone.
c. What are osteoclasts? Osteoclasts are endodermal lineages that are
recruited to the bone following vascularization.
d. What lineage are osteoblasts? Mesoderm or neural crest derivative
2. Bone diseases
a. Defects in osteoblast lineage causes you to make too little bone and
diseases such as osteogenesis imperfecta. Causes bowing of the legs
because the bones arent strong enough to support the weight of the
individual.
b. Diseases in osteoclasts do what? Diseases in osteoclasts causes you to
have too much bone and causes osteopetrosis. Causes bone to be too
dense and can break easily.
3. Dermal Bone growth in the Skull
a. What are the 5 predominant bones of the skull? (2)Frontal, (2)
Parietal, Interparietal.
b. Where does bone growth in the skull occur? At sutures.
i. What are the different sutures?
1. Coronal, Lamboid, Metopic, Saggitall
c. What is craniosynostosis? A premature fusion of the bones in your
skull , the calvaria.
i. When you are small, your head is undergoing growth and the
skull needs to expand along with bone growth. Skull is not
fused, but then it becomes fused after growth. Timing of fusion
is very important.
ii. Anteriorally, the first bony element are the frontal bones which
are paired. Just posterior to that are 2 paired parietal bones.
Most posterior is interparietal bone. 2 Frontal, 2 Parietal, 1
Interparietal. Early in development, they are not fused. The
metopic suture (between the two frontal bones), Lamboid
(between parietal and interparietal), and Coronal (between the
frontal and parietal) and saggital (between parietal and
parietal).
iii. What is the requirement for craniosynostosis? The fusion of
ANY one suture. It doesnt require all sutures to be closed. This
causes pressure within the skull because of the pressure
resulting from bone growth.
d. What are the causes of cranialsynostosis?
i. In where it is known, craniosynostosis is caused primarly by
disruptions to fgf signaling. This occurs through mutation of
one of the three fgf receptors or mutation of the transcription
factor Twist that mediates fgf signaling.
e. What type of mutation is present in human craniosynostosis?
i. All the mutations are activating mutations. That render the
receptor overly active. It is fgf signaling that is uncontrolled
that causes it.
4. Muscle Development
a. Myogenesis: generation of muscle
b. Individual unit of a muscle is a myofiber which is a multinucleated
cell. Myofibers are contractile, provide force for the muscle, and are
multinucleated cells.
i. Contractility is governed by several different proteins
including myosin heavy chains.
ii. Differentiated myofiber characteristics: multinucleated and
has myosin heavy chains which are contractile.
c. Muscles come from the dermamyotome of the somites. All of the
muscles in your body comes from paraxial mesoderm that generate
somites.
d. Conversion of myoblasts into muscles
i. What are myoblasts? Muscle progenitor cells
ii. Myoblasts start expressing two basic HLH T.F. cells. MyoD was
the first one that was discovered and MyoD is sufficient for
activation of myosin heavy chains.
iii. MyoD is sufficient enough to drive the differentiation into a
muscle cell.
iv. MyoD-/-: Muscles are normal. Its not necessary because there
is another bHLH T.F. called Myo5 which is expressed
overlapping with MyoD.
v. Myo5 -/- normal muscle
vi. MyoD;Myo5 -/- No muscle development. This means that
these two transcription factors are functionally redundant,
similar to Hox genes. Either one on its own will allow
differentiation into muscle cells.
vii. As they begin expressing MyoD and Myo5, will start
proliferating.
viii. What pathway is important in muscle development? Fgf
signaling is necessary to maintain the myoblasts in an
undifferentiated state, or proliferative state.
ix. Fgf-/- in myoblasts: will undergo diffentiation and exit the cell
cycle before fusing.
e. Cells that are proliferative are not aligned. We can see this from the
experiment with labeled radioactive thymine where the fgf signaling
is present, the myoblasts are random. When fgf signaling stops, now
they are elongated and align with one another.
f. You stop proliferating, and then you align.
5. Differentiation-Terminal differentiation
a. MyoD and Myo5 directly target another bHLH called myogenin.
b. What is the function of myogenin? Myogenin is a bHLH transcription
factor that is necessary for final differentiation and expression of
myosin heavy chain.
c. Myogenin-/- You still have undifferentiated muscle cells. The
proliferation of the myoblasts is normal. Youve made cells that should
have become muscle but the cells did not undergo the terminal
differentiated needed to become muscle.
d. Myostatin-/- massive expansion of muscle. Myostatins function is
to block muscle differentiation. It is TFG family member. However,
they have a lower survival than heterozygotes.
e. What can myostatin blockage be useful for? Muscular Dystrophy
6. Myoblasts form myotubules
a. What is the fusion of myoblasts to form myotubules initiated by?
Meltrins. What are meltrins? Fusogenic proteins similar to bindin.
b. What is the relationship between meltrin and myogenin? They are
coexpressed, meaning that meltrin is only expressed in cells that
express myogenin because meltrin is a target of myogenin.
c. Meltrin overexpression: Causes myoblasts to fuse inappropriately,
showing that meltrin is sufficient for myoblast fusion.
7. Alignment of myotubules to form myofibers
a. Wnt11 drives the appropriate alignment of cells. The function of
Wnt11 is to orient myotubules after meltrin has fused them.

11/25

1) Vascular Tube Formation
a. We have to make a lumen. The last step of vascular development is
tube formation, it has to be able to carry blood and for that it has to be
hollow and this happens through tube formation and fusion of
endothelial vacuoles.
i. Vacuole fusion: Tube formation during vaculogenesis requires
endoderm.
1) What are the two different types of vaculogenesis?
Intracellular and intercellular fusion.
2) What is intracellular fusion? Intracellular fusion is
fusion within the cell and it is necessary to increase the
space of the intracellular space.
3) What is intercellular fusion? Intercellular fusion is
fusion between cells and it makes the lumen.
4) These two types of formation is necessary for lumen
formation and requires signaling from endoderm.
5) What is the endoderm responsible for? Migration of
progenitors and formation of the tube itself.
b. What is vasculogenesis? Initial generation of blood vessels that
requires Hemangioblastsa that migrate on the endoderm and require
Vegf.
c. What is angiogenesis? Remodeling of the network generated via
vasculogenesis and generation of capillary beds. Endothelial cells
need endoderm, Wnt & R-spondin to maintain Vegf for sprouting.
d. How does the lumen form? Via fusion of vacuoles
2) Teratology
a. What is Eco-devo? The study of how the environment influences
development.
i. Fishes: sex determination is determined by density of other
fish/temperature
ii. What is teratology? How the environment can disrupt
development. Influencing development by being detrimental.
b. What is a teratogen? A teratogen is an environmental agent that
causes developmental defects
i. What does thalidomide do? Children born had severe limb
defects. It was prescribed to pregnant women to prevent
morning sickness. Thalidomide caused limb defects.
3) Sensitivity of embryonic organs to teratogens
a. In any time period, there are important organs that could be
influenced by teratogens. CNS forms earliest and longest, which is
why CNS is one of the most commonly disrupted organs by
teratogens.
4) Ethanol
a. What is the most common teratogen? Ethanol
b. What does ethanol cause? FASD-Fetal Alcohol Spectrum disorders
which refers to any deleterious effect alcohol has on an unborn child.
i. One type of FASD is? FAS is a clinically diagnosable disease and
the most severe type of FASD.
ii. What does diagnosis of FAS consist of? Specific facial features
and severe neurological impairment.
iii. What are the specific facial features? Smooth philtrum, thin
upper lip, underdeveloped (small) lower jaw, and small head
(hypoplasia of craniofacial skeleton) and shortened palpebral
fissure length.
iv. What are the neurological effects? Sulci and gyri are greatly
reduced and the cortex is smooth (lissencephally). The brain is
often smaller (esp cerebellum) and the major commissures are
disrupted, such as the corpus callosum.
v. With a child that has FAS what is a characteristic of the corpus
callosum? White matter fibers that pass through the corpus
callosum are reduced in number. Decrease in projections
across the corpus callosum.
c. The face as a window to the brain
i. Relationships between the face and the brain
1) Philtrum scale: Deep philtrum to very shallow philtrum.
The philtrum associates with the thalamus (an
important relay center of the brain). The philtrum
measure correlates wit thalamus size.
2) The size of your palpebral fissure length (size of eye
opening) is correlated with smaller size of the corpus
callosum (less projections).
d. How does alcohol cause FASD?
i. Apoptosis: particularly of neural tissues, presumably because
of its metabolic demands. Losing specified neural tissue
through apoptosis.
ii. Inhibition of L1: L1 is a homotypic adhesion molecule similar
to cadherin important in the development of neural fiber
tracts. Inhibition of L1 might play a role in the disruption of
cortical tracts. As the amount of alcohol increased, you
decrease L-1 Mediated cell adhesion.
iii. Can increase reactive oxidation species that conduct apoptosis
5) Variability in FASD
a. Gene-environment interactions.
i. Concordance rate in Identical twins for FAS (100%) and
Fraternal twins is 63%.
ii. What are the genes and what might they be doing? You can
conduct genetic screens to find the genes that generate
susceptibility to alcohol.
6) Ethanol interacts with pdgfra
a. Ethanol treated Zebrafish pdgfra interactions with Ethanol.
b. What is haploinsufficiency? One copy of the gene is insufficient.
c. If youre exposed to ethanol, you need two copies of pdgfra. If you are
not exposed to alcohol, you only need one copy of pdgfra. Pdgfra is
important in the migration of neural crest cells.
d. Untreated pdgfra has the same phenotype in both the +/+ and the +/-
(haploinsufficient) genotype
e. What is the function of pdgfra? Alcohol increases cell death. Pdgfra
protects against alcohol exposured apoptosis.
f. Synergistic-not additive
g. Pdgfra is a RTK which binds ligand and phosphorylates downstream.
One of the things it phosphorylates is PI3K which regulates cell
migration and cell survival.
h. PI3K signaling is sufficient to guard against alcohol apoptosis. If the
PI3K is blocked in a normal situation (without alcohol) you have
apoptosis. Pi3K is necessary to block apoptosis.
7) What is the relationship between Choline and FASD?
a. Choline is a nutrient that is necessary for making the cell membrane.
Choline supplementation has been demonstrated to improve FASD by
having fewer neural defects. Pregnant women who drink have
lowered levels of nutrients including choline.


12/2

1. What are endocrine disruptors? Molecules that alter endocrine signaling.
They are also known as environmental estrogens, and they can alter primary
and secondary sexual characteristics.
2. What are the two types of endocrine disruptors?
a. Agonists: activate the endocrine system. An example of these are the
environmental estrogens which activate the estrogen pathway. An
example is DES, which activates estrogen pathway. It was prescribed
to pregnant women and caused defects to sex organs and caused
increase in cancer.
b. Antagonists: block endocrine system. An example of this is DDT which
blocks hormone activity.
3. Most of these agonists are components in ________ which is associated with
elevated ________ __________.
4. As the use of environmental estrogen increases, you have increase risk in the
cancer of ____________ _______________ and defects of ____________ _____________.
a. What are the defects? Reduced sperm count, defective testicles
(Hypospadia).
b. Cancer of sex organs: testicular and breast cancer.
5. What is the effect of the environmental estrogen on inheritance?
Reproductive defects can be transgenerationally inherited.
a. What are transgenerational defects? Transgenital defects are defects
seen in the generations that are not exposed to the environmental
estrogen.
6. How do you generate the transgenetic effects? Epigenetic changes such as
DNA methylation which can turn genes off.
7. Sensitivity to estrogen is likely to be _________________.
a. Gene-environment effects have a similar effect with environmental
estrogens as they had with alcohol.
8. Regeneration of Missing parts- can we replace missing parts?
a. What is the champion of the regeneration world? Planaria
b. What is the key step in regeneration?
i. Formation of blastema: group of undifferentiated cells at the
injury site which replaces the differentiated cell type
ii. Posterior blastema makes: tail
iii. Anterior blastema makes: head
c. What are the proliferating cells in planaria? Neoblasts
i. What does BrDU do? Labels proliferating cells.
ii. Neoblasts in the planaria are seen throughout the body.
1. What are neoblasts? Cells that can make cells.
2. Do the neoblasts give rise to the blastema? 3 pieces of
evidence
a. Planarai was cut and then labeled with BrDU.
BrDU labels proliferating and undifferentiated
cells and it was shown that the blastema
contained BrDU labeled cells, therefore the
blastema contained neoblasts.
b. Irradiated planarians cannot regenerate because
you kill proliferating cells. Why specific to
proliferating cells? They have to maintain
chromosome integrity which doesnt matter in
differentiated cells.
c. Isolating and transplanting the cells that have
taken up BrDU rescues regeneration- partially
restores the ability to regenerate.
d. How are neoblasts regulated?
i. What makes a neoblast?
1. This is tested by a forward genetic screen where many
genes are wiped out and then the phenotype is seen.
2. Wi genes in planarians (Piwi in vertebraes)
a. Wi 1 & Wi 2 are RNA binding proteins and
regulate gene expression and are expressed by
neoblasts.
i. Irradiation of planariagetting rid of its
neoblast nature also are defective in wi1
and wi2.
b. Function of wi1 and wi2 maintains stem-
ness or the ability of a cell to be pluripotent.
ii. How do we maintain the polarity of the embryo?
1. Wnt pathway is responsible for this.
2. B-catenin-/- generate two heads
3. Regardless of where the amputation occurs, blocking
Wnt signaling results in anterior duplication.
a. Wnt has an anterior to posterior gradient. Wnt is
highest in posterior and anteriorally, you have
the highest concentration of Frisbee which
inhibits Wnt. Therefore, Frisbee anteriorally and
Wnt posteriorally. In the second half of the cut
planarian, the expression of Notum works like
Frisbee, inhibiting Wnt signaling in the anterior
half of the blastema. Notum is only expressed in
response to injury and Frisbee takes back its
place after the blastema is regenerated.

12/4

1. Unlike Humans, Zebrafish can regenerate _______ ___________
a. In Zebrafish, cutting out a small piece of the heart generates a
blastema from which the new heart tissue is generated.
b. Does this involve the de-differentiation of already differentiated cells
or new cells forming the tissue?
c. Injury reactivates GATA4 expression in cardiomyocytes
i. In adult heart- there is no GATA4
ii. GATA4 is expressed embryonically during the differentiation of
cardiomyocytes that generate the heart.
iii. What does injury do? Injury reactivates GATA4 and other
developmental genes such as Nkx2.5 and hand2 and tbx20.
iv. The developmental program is __________. Reactivated.
d. Is the regenerated portion of the heart due to the expression of the
cells that have expressed GATA4?
i. Lineage tracing: Following the lineage of GATA4.
ii. Cre is driven by GATA4 promoter. Cre recognizes LoxP sites
and we have the recombinase under the activation of GATA4.
Fluorescence switches from red to green when GATA4 is
active. After activating Cre, you have the cells expressing green
fluorescence in the GATA4 lineage. Non GATA4 expressing cells
will remain Red. Beta actin then drives the cmlc and gata4
generation. The GATA4 lineage does contribute to the
regeneration of the heart and we have regenerated the
developmental program.
e. Where are these cells coming from? New cells that differentiate or de
differentiate?
i. Experimentally: You can use CMLC (specific cardiac gene in
differentiated cardiac tissue).
1. Injure heart turn on Cre What happens to lineage?
The differentiated cardiomyocytes are de differentiating
and reexpresing developmental genes and undergoing
proliferation back into cardiomocytes. Not through a
stem cell population, but a de differentiation.
f. Do humans have any capacity of heart differentiation?
i. Cold-War elevated the level of radioactive C14. People born at
specific times have specific level of C14. You can measure the
level of C14. There is turnover of cardiomyocytes in humans.
People born before WW2 have a higher level of C14 and people
that were born after have a lower level of C14.
2. Transdifferentiation: suggests genomic equivalence
a. Genomic equivalence means?
i. Every cell in your body has identical DNA
b. What is transdifferentiaton?
i. Dedifferentiation of one cell and redifferentiation into another
cell type.
c. Stem cells
i. Pluripotent cells
d. Reprogrammed cells
i. Not a stem cell originally, but want to make them stem cell like
e. Embryonic stem cells
i. Generate any cell type in the embryo and are pluripotent and
are capable of cell renewal (can generate themselves).
f.