• There are about 10 million species on Earth.
• Researchers have selected model organisms to study all species (i.e.: zebra ﬁsh, mouse). These
organisms are used to study our cells.
Basic Process of multicellular organism Development.
• Broken down into four basic components.
• All occur in parallel and without global supervision (occurs due to genetic programming).
• Understanding how the genomic information encodes something so complicated.
Human ﬁgure details visible in human embryo after just 8 weeks. Toes, ﬁngers, most organs and the
brain are visible. Until week 38 (around birth), proliferation is mostly what takes place.
• 8 weeks: ~35 mm (Advanced organogenesis). At 38 weeks ~50 cm.
• Adults have 6x10 13 cells which originated from a single zygotic cell. Lining up these cells in order would
wrap 75 times around the earth.
• There are ~250 different cell types (i.e.: neurons, muscle cells, adipocytes). Thus, cell specialization
• Formation of germ layers. Extremely important step to generate 3 types of cell:
1. Endoderm: Precursor of gut, liver, lungs, pancreas.
2. Ectoderm: Forms the outer, protective layer called epidermis and
3. Mesoderm: Precursor of muscles, skeleton, kidneys and connective
tissue. • Endoderm forms the inner tube that connects the two ends. Organs grow out of it.
• Forming the three cell types from the ﬁrst ball of cells is the ﬁrst step of diversiﬁcation.
• Gastrulation (formation of mesoderm).
- Involves epithelial-mesenchymal transition. This also occurs in cancer (cancer cells undergo this
transition to migrate around the body).
- Cells become more pyramidal in shape and move within the embryo. Cells lose their lesion to
- Snail induces epithelial cells to become mesoderm by repressing E-cadherin (which links cells
together in adheren (tight) junctions). Cells then lose lesion and change fate.
• Second way to create cellular diversity: Sequential Induction
- Two different tissues (A and B) adjacent to one another
will interact. Within tissue A, tissue C will form because
of this interaction. Tissue C can then interact with
tissues A and B to form tissue D. The more tissues
there are, the more possibilities for interactions.
- This occurs in the eye (and a few others places too).
Two individual, adjacent cell layers have distinct
identities until retinal layer interacts with the
pigmented epithelium (outer layer). These cell types do not form individual layers but are rather,
combinations of molecules that are expressed.
- Interaction between cell layers allows for differentiation into more layers.
• Changing the fate of your neighbors
- Group of cells sends signals to neighboring cells to change their fate.
- Cells send an inductive signal. This ‘induces’ a fate. Cells receiving the signal, will interpret it, and
turn on a new cellular program which will change their identity.
- An example: Drosophila wing starts off as a pouch of cells that unfolds to form the wing. The midline
(separates ventral and dorsal sides) of the pouch will form the edge of the wing. Wg is a morphogen
and will travel to neighboring cells to turn on dll. Cells that are surrounding the midline will begin to
turn on dll. Vg is expressed in a wider region between cells are more sensitive to Wg signal in this
case. Same molecule secreted from midline and neighboring cells will turn on both dll and Vg but
then those cells further away will not active dll, only Vg. This creates 3 cell types (those that express
all 3, those that express only dll and Vg and those that expressed only Vg). Morphogen
- Principle of a morphogen: one inductive signal creates multiple (in this case, 3) cell fates.
- Acts at different concentrations. At high concentration, signal will drive a certain information. At
medium concentration, it will drive another kind of information etc.
- Morphogen itself does not determine fate, it is the way in which the cells receive it.
- Molecule is secreted from a source.
- It is time dependent. The longer the cell is exposed to a morphogen, the more it has a chance to
move on in its interpretation of the signal.
- Usually morphogens are sticky.
- Can be modiﬁed and regulated by different mechanisms (i.e.: ECM or proteins surrounding the
neighboring cells can exert an inﬂuence).
- Induction over a short range may take place ﬁrst. Proliferation can then occur so that the range of
action of a single morphogen can be very large.
- French ﬂag model explains how morphogen acts (a range of concentrations where the cells act).
- Different systems can receive the same morphogen signal. It is not the morphogen that
determines the fate of the cell. It is the cells’ method of interpretation. This allows for a single
morphogen to be used in many systems.
• Two ways to create a morphogen gradient:
1. Classical way to create a morphogen gradient: source inducer diffuses. A gradient moves away from
2. Inducer can be expressed everywhere and an inhibitor (in the form of a gradient) acts. Close to the
inhibitor, the inducer is unable to act. There exists a gradient of inducer activity. This occurs in ﬂies.
• The result is the same for the both methods. • Some important signaling pathways for embryonic development:
- Wingless (in ﬂies) and Int (integration of a virus in vertebrates) together become Wnt.
- Some were discovered in the embryo.
• Much information we know from the pathways comes from a single screen where mutations affecting
the patterns of the ﬂy larvae (from Drosophila) is studied. Mutations are induced, and the patterns of
results are studied.
• The pathways being studied know are for Cancer and other diseases.
• Posterior end has naked cuticle while anterior end is covered in bristles. They have a speciﬁc
pattern and an identity for each segment of the larvae.
• The mutations being generated would generate only 3 kinds of phenotypes:
1. GAP gene: a whole series of segments were missing
2. Pair-rule gene: either even numbered segments or odd numbered segments were missing.
3. Segment polarity gene: Correct number of segments but polarity is wrong. • About 15 patterning genes in 3 categories were found.
• A mutation was then induced in the mother. Additional 8 genes were found that are involved in tail and
anterior head formation. These gens are referred to as the maternal contribution.
• The reason why these three categories exist is because there is a sequential event that occurs. Steps:
1. The polarity of the embryo was deﬁned. Morphogen creates a gradient to do so. At high
concentration, the hunchback gene is turned on.
2. Cells respond to a gradient to express three cell types.
3. The three types interact to act in sequential induction.
4. Response to primary segmentation. Two genes being activated (one is even skipped the other is
fujitso). Alternative segments express one of the either.
The Canonical Wnt Signaling Pathway
• Often related to hair phenotypes because they study the cuticles.
• Without this pathway, there is no signaling at the membrane. This leads to formation of a complex that
is controlling beta-catenin that is the key molecule in the pathway.
• CKI alpha and GSK phosphorylate beta-catenin.
• They then enter ubiquitination and degradation pathway.
• TCF then binds Groucho which recruits a repressive complex that is essentially shutting down genes.
Wnt binds receptor frizzled and co receptor LRP. This decomposes the built complex. Beta-catenin
cannot be phosphorylated anymore and it accumulates in the cytoplasm, translocates to the nucleus
where it binds TCF and displaces Groucho. This turns on transcription of a series of genes needed in
proliferation. • In vertebrates, many Wnt have been found (1-5). They are linked to different diseases such as
• In Wnt 4, the genes are for pseudo males. Thus, females is not the ground state. Instead, a single
drives the gender towards female.
• Wingless and Int are the same thing. Wingless is the molecule that was identiﬁed in the ﬂy. In
vertebrates, there were other groups of people that identiﬁed Int gene. They later realized these two
molecules were the same in both species. So the term ‘Wnt’ was created to encompass both. In ﬂies,
some people perform the term wingless.
• Canonical pathway: refers to the dominant pathway, the most well known / best characterized pathway.
Once a second pathway downstream was identiﬁed, the original one adopts the term ‘canonical’.
Sonic Hedgehog and BMP
• Act as morphogens in the neural tube.
• The spinal cord / neural tube is visible in the early embryo in green. When cross sectioned, the image to
the right is obtained. The cells are proliferating and will eventually give rise to different kinds of neurons.
In order for the neurons to obtain their distinct identity types, they rely on the morphogen sonic
• Hedgehog was identiﬁed as a mutant in the ﬂy ﬁrst. Later, three were found in vertebrates (and labelled
with respective to the video game); sonic hedgehog, indian hedgehog, desert hedgehog.
• Sonic hedgehog is the most well known and best characterized of the three. It is also the most active of
the three. It is essential in the patterning of the spinal cord.
• During development, a cord of tissue runs all the way down below the spinal cord, the noto cord. This
tissue expresses sonic hedgehog. It ﬁrst signals to the ﬂoor plate (lower part of the spinal cord) and
turns itself on in this tissue. It is a transfer of expression. Sonic hedgehog then starts to signal from the
dorsal side, in the ventral direction. It then provides the identity to different cells.
• Closer to the ﬂoor plate, there will be a higher concentration of sonic hedgehog, inducing one type of
fate. Lower down, different fates emerge according to concentration levels. This is seen by the various
colors in the diagram below. These domains have different combinations of molecules. Sonic Hedgehog Signaling Pathway
• Double system: activation and repression occur simultaneously. This allows precision.
• The challenge is to generate clear borders between different cell types. The double system allows for
this to be possible.
• Ground state: refers to non activated state. Absence of ligand.
1. Patch I represses the Smo receptor.
2. PKA phosphorylates Gli3.
3. Gli3 is phosphorylated, cleaved by proteolysis. This generates an inactive repressor molecule.
4. Gli3 translocates to the nucleus and binds target genes to actively repress them.
• Repressor function allows for repression of genes.
• Sonic hedgehog represses its receptor and then becomes inactive to represses Smo. This is a double
negative that turns positive.
• Gli3 becomes an activator from being a repressor.
Once target genes are activated, the amount of sonic hedgehog will determine how much activation
occurs. It is therefore a gradient response. The ligand does not reach far distances to the tissue there
remains in ground state.
• Cyclopamine: Smo repressor. System remains inactive, despite the presence or absence of sonic
hedgehog. Acts like sonic hedgehog mutant. The whole pathway is shut down by this molecule.
Cyclopia was born as a cyclone because its mother ingested the plant that contains a cyclopamine
molecule. This molecule is the inhibitor of Smo.
• Sonic Hedgehog in the embryo, acts to specify the midline to differentiate this line in the body. • A cyclone is the fusion between in the eyes because the midline does not form. In a sonic hedgehog
mutant, the midline does not form properly and different organs will fuse together (i.e.: one kidney).
• Mutants of sonic hedgehog can lead to loss of Shh signaling which causes holopresencephaly
(variable penetrance). There is a range of severity that can occur and the most dramatic cases are
• Acts as sonic hedgehog’s partner. Expresses from the dorsal side towards the ventral side. It is the
inverted signaling pathway.
Less well characterized than sonic hedgehog’s pathway.
• Roof plate expresses BMP and signals downwards to turn on combinations of transcription factors in a
concentration dependent manner to provide different identities to different cells along the ventral axis.
• Is involved in spinal cord patterning. Re used in different systems.
• Canonical Pathway:
1. BMP binds its receptor (formed of a dimer of receptors 1 and 2). These molecules dimerize upon
2. Receptor 2 will phosphorylate receptor 1. This is the activation trigger.
3. Phosphorylation of receptor 1 recruits Receptor-regulated Smads (R-Smads): Smads 1, 5 and
8. There are three different proteins that carry out the same function.
** Phosphorylation can be repressed by inhibitory Smads: Smads 6 and 7. This allows a break in
the pathway. 4. Smads 1, 5 and 8 translocate down to recruit co-Smad: Smad 4 which binds to anyone one of
the three forming a complex.
5. This complex translocates to the nucleus, binds target genes and turns them on in a graded
manner. The more BMP, the more activation of the pathway.
• Non canonical pathway:
1. Binding of XIAP activates tyrosine kinase TAK1.
2. Different pathways are activated. TAK1 activates the MAPK pathway (MKK), JKN pathway and
3. Those pathways are involved in different target genes. The response of these genes varies.
Controlling Cell Fate From the Nucleus
• Combination of gene regulatory proteins (transcription factors), which are often activated by
inductive signals, will determine the fate of a given cell.
• Smads are transcription factors.
• Gene regulatory proteins bind enhancers (DNA sequences), which can be located upstream or
downstream of the gene. They bind speciﬁcally based on their identity. They act as on the on switch to
turn on transcription of the gene.
• Type of transcription factors, ﬁrst identiﬁed in drosophila.
• The genes are all found in a cluster, lined up one after another. At one extremity, they are expressed in
the anterior part of the body and the opposite on the other. There is a correlation between the position
along the chromosome and the expression domain in the body.
Well conserved in bilateria.
• Important for organizing the pattern of body tissue. Provide identity to the tissue they are expressed in.
They tell the cells where they are in the body.
• Antp and Ubx are two types of Hox genes often seen mutated.
• In a wild type ﬂy, there are 3 body segments. In the second segment, the wings are present and in there
are haleteres in the third segment. Haleteres are used by ﬂies to maintain balanced.
• Homeotic transformation: change cellular identity towards a different fate.
- Ubx mutant develops a second set of wings in the third segment as opposed to haleters.
- Ubx over expression in the second segment causes wings not to be developed. Instead, haleters
develop in their place.
- Antenapedia (Ant) cause ﬂies to develop legs in the place of antennas. • Hox genes are well conserved among species. In the mouse, there are 39 Hox genes found in four
clusters (on 4 different chromosomes). In vertebrates, the clusters were duplicated twice such that four
• Colinearity: the alignment of genes along the chromosome correspond with their expression pattern
along the embryo. Unique pattern provides identity.
• HoxB2: Expressed in the posterior level but does not have a sharp boundary.
• HoxB4: Expressed in the posterior level but has abrupt stop and sharp boundary.
• Removing HoxA10 (normally expressed in the lumbar region) and expressing it all along the vertebral
column: the whole spinal cord becomes lumbar. The vertebrates do not form ribs. Thus, each segment
is a read out of its gene. The reverse experiment (inactivation of HoxA10/C10/D10 which all perform the
similar function - triple knockout): lumbar and sacral vertebrate becomes thoracic like.
• There are two ways to create diversity:
1. Before cell division
- Asymmetric cell division. The determinant (something that will inﬂuence the fate of the cell)
is only exposed on one side of the cell. Upon division, only one of the two daughter cells will
express the determinant.
2. After cell division
- Post division, one of the two daughter cells is inﬂuenced by its environment.
• One example: Speciﬁcation of sensory mother cell (one cell that is speciﬁed to become the precursor
to the whole sensory organ).
- Performed by interaction of two cell post division. - After several divisions, the precursor cell develops a socket cell (holds the bristle) and a shaft cell
(the bristle). Upon the second cell division, one cell normally dies. The other divides into a sheath cell
(protects the neuron) and a neuron (links organ to CNS).
- This occurs by way of lateral inhibition. All of the cells of the ectoderm compete against one
another. Each cell does not want its neighbor to become a sensory mother cell. They all repress each
- At some point, equilibrium is broken. One cells wins over its
neighbors. Once the neighbors become weaker, they shift their
balance so its other neighbor can win. This generates a pattern of
nicely spaced out, separated cells that lock their fate in the
- The winning cells will leave the ectoderm and become the
sensory cells. The losing cells do not die - they simply accept the
- Two cells are equivalent bus a transient bias creates a small
asymmetry where one cell becomes better than the other. A
system comes along to amplify this asymmetry. By one cell being
repressed, it creates a positive feedback loop.
- The Notch pathway is what is responsible for shifting the balance at the molecular level. - Delta does not diffuse to multiple tissues. It remains in place. Only cells that are adjacent interact with
one another. Delta activates Notch in one cell and this creates a domino effect of negative
regulation. The cells compete to a point where one delta is stronger than another cell which will
prevent the activity of the other cell.
- Diagram above is a more detailed image of the pathway.
- Cis inhibition of Notch (delta prevents Notch activity) is an all or nothing response. When Delta is in
a cell, it binds Notch in the same cell (thus, cis) to prevent its function. This occurs in both cells.
- In trans (between two cells), Delta acts as an activator. Delta goes to the membrane and binds Notch
on the other cell. This ligand-receptor binding turns on transcription as a graded response (as
opposed to cis which is an absolute response). The more delta, the more Notch response.
Notch Signaling Pathway
• Notch signaling occurs by interactions between neighboring cells.
• Notch and Delta exist on two neighboring cells.
• Signaling Pathway:
1.Notch is glycosylated and processed by protein cleavage so that it may be re attached to the cell
2.Notch binds Delta and a conformational change in the protein occurs that induces cleavage of the transmembrane domain. TACE Proteases cut at one position and gamma-secretase cuts at a
different position in order to release the NICD (Notch Intracellular Domain) into the cytoplasm.
3.NICD is a transcription factor that translocates to the nucleus. NICD transcriptional factor displaces
the co-repressors (CoR), binds CSL transcription factor and brings in co-activators (CoA).
4.Turns on transcription of target genes (typically Hes and Hey gene families)
The signal from membrane to the nucleus usually goes through several phosphorylation steps.
However, in this case, it is a piece of the cell receptor’s intracellular domain that directly enters the
nucleus and performs the required function.
• The extra cellular domain of Notch is released by protein cleavage and remains bound to Delta. This
can be problematic because the system is blocked by the presence of this interaction. The
degradation of this complex is important.
• Mindbomb is a ﬂy mutant that was characterized by researchers. It was ﬁrst discovered and then only
• This system is involved in a number of pathways.
• The speciﬁcation of the sensory mother cell occurs by lateral inhibition where a cell ‘wins’ over its
neighbors, generating a repetitive pattern of equally spaced cell types.