Download The retinal neuroepithelium contains retinal progenitor cells that

ANAT 416
Dr. Cayouette
February 14, 2012
Lecture 11
ANAT 416
Lecture 11 – Eye Development
Dr. Michel Cayouette – February 14, 2012
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not all of the text contained in the lecture slides will be reproduced here.
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Lecture Outline
Part I: Development of the Eye
Part II: Development of the Retina
Part III: Mechanisms of cell-fate specification in the retina
Part I: Development of the Eye
The Eye Develops from Embryonic Day 9.5 in Mouse
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The eye develops from about embryonic day 9.5 in the mouse and in humans it starts from the
third week in gestation. It’s an early process in humans and is in mid-embryogenesis in mice.
At day 9.5 can see bulge on the head and can recognize as the eye forming. From about E11.5
can see the eye clearly on the head of the mouse.
The Eye Develops From a Series of Inductive Events
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Since the retina develops from the neural tube, it is part of the Central Nervous System (CNS).
The neural tube develops from the neural plate. The neural plate is open at the beginning of
mouse development and closes to form the neural tube which develops in the CNS.
Anteriorly, there are two expansions in the neural tube:
o Optic cup – budding of the neural tube
o Lens placode - in front of the optic cut and forms the lens. The placode invaginates,
pushing the optic cup inside so
that it forms a two-layer
structure:
 Anteriorly is the retina,
while the retinal
pigmented epithelium
(RPE – shown in black
in the diagram) is more
posterior.
 These two structures
come together and form
the light-sensing part of
the eye, which is the
Figure 1: Formation of the Eye
function of the retina.
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Lecture 11
o RPE – multiple roles:
 Trophic support: delivers nutrients to the retina.
 Structural support: removes oxidative damage caused when light hits the retina.
Note, the cornea and lens (as well all other parts of the eye) are not derived from the neural tube
and thus, are not derived from the CNS.
Eye development
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Using Scanning Electron microscopy (to obtain a 3D
perspective of image), you can look at a section
through the eye of an embryo. Here, you can see
budding of the neural tube and optic cup formation.
Note the lens placode forms a thin layer on the
neural epithelium, and then thickens.
The cells in the periphery will form the developing
lens. These cells invaginate, which pushes on the
optic cup to form anterior and posterior portions.
Therefore, the movement of cells and their
distribution in a 3D environment lead to the
formation of the optic cup, and other eye structures.
Figure 2: Eye Development
The adult eye
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The list on this slide is mostly for your own information. Important
to know what the eye looks like.
The retina is the only light sensitive area of the eye. Its main role is
to transfer light to the CNS.
Figure 3: Adult Eye
Also have cornea, lens, iris, etc.
Part II: Development of the Retina
The Retinal Neuroepithelium contains Retinal Progenitor Cells (RPCs) that Divide to Generate
Different Retinal Cells
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The RPCs are also known as the retinal progenitor cells as they give rise to the retina. These cells
proliferate and differentiate into different types of neurons and different retinal cell types.
The retinal cells in human and mammals do not continue to proliferate throughout life, which is
why we can’t regenerate photoreceptors in certain diseases, and thus, should not really be called
stem cells.
In vivo live imaging of dividing retinal progenitor cells
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Two videos in zebrafish: you can see these cells divide and expand dramatically in the first few
hours of retinal development.
Zebrafish are good model organisms for studying eye development because they are transparent
and can be immobilized in an agarose gel to observe retinal development in real time.
Looked at retinal progenitor cells dividing (labeled with green fluorescent protein, or GFP)
Arrows indicate mitotic cells at the apical surface.
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Lecture 11
The retinal neuroepithelium contains retinal progenitor cells that divide to generate different
retinal cells – part II
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Usually when look at other epithelia, the apical surface faces the lumen.
o Example, in the brain, the apical surface of ventricles faces the lumen.
However, in the eye, the apical surface faces the outside. The lumen is the neural tube.
After the invagination event, the apical surface will be closely opposed to another apical surface
– so two apical surfaces are facing each other. When look at eye, may be tempted to say the
apical surface of the retina is facing the lumen so its inside the eye, but it’s not due to folding of
the retina.
Development of the rodent retina spans a period of two weeks
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Development of the mouse retina takes about 2 weeks from embryonic day (E) 9.5-10 to
postnatal day (P) 7.
If focus on retina, and look at E18, see one layer of neuroblast progenitor cells, NBL, (another
term for progenitor cells) and a retinal ganglion cell layer. These are neurons.
If look at later stages, such as P4, you can see 4 distinct stages:
o ONL – outer nuclear layer
o Thick layer in middle
 NBL – neuroblast layer
 INL – inner nuclear layer
o GCL – ganglion cell layer
By P7, have final structure of the retina, the ONL, INL, and GCL.
Basic organization of the vertebrate retina
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There are three distinct layers in the retina
The ONL contains the cell bodies of the photoreceptors, the
rods and the cones.
o In this layer, light is transformed into a nervous impulse.
o Photoreceptors contain photo pigments:
 Rods – rhodopsin
 Cones - opsin
o Rods are useful in night vision, as they are very
sensitive, but not as good at differentiating details.
o Cones are useful in daylight and fine precise vision –
looking at colors, reading, etc.
The interneuron cells (horizontal, amacrine, bipolar cells)
Figure 4: Vertebrate Retina
integrate the signals received by the photoreceptors, for
example, is it high or low contrast? Is it textured or smooth?
o This layer also contains the cell bodies of Muller glia, which are the main cell type of the
retina (spanning the entire length). These cells provide structural and trophic support.
The retinal ganglion cell (RGC) layer contains RGCs which are the only projection cells of the
retina. These neurons send an axon out of the eye to the brain. The axons of multiple RGCs form
the optic nerve, which projects to the lateral geniculate nucleus (in humans), and then on to
higher visual centers. This happens very quickly.
The retina is used as a model system to study nervous system development
o ie, when we ask the question: how are different cell types generated from a pool of stem
cell or neural progenitor cells?
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Lecture 11
o Why is it a good model?
 It is a simple structure with three distinct layers
 There is a manageable number of cell types (only 7)
 Versus cortical progenitors with hundreds of different cell types
 In addition, we have cell-type specific markers for each of these 7 cell
types
 Immunohistochemistry – technique which uses antibodies to identify
proteins expressed in these cells. For example, only rods contain
rhodopsin, so we could detect the presence of rhodopsin with an antibody
conjugated to a fluorescent molecule.
 The retina easy to access, as opposed to the brain (would need to drill into the
skull, etc).
 There are two eyes, so one can be used as a control.
Example of histological staining:
o In the retinal section, see photoreceptor cells stained with protein specific to
photoreceptor cells. Also see bipolar and amacrine cells stained with different, cell typespecific marker.
Part III: Mechanisms of Cell-Fate Specification
How are the different retinal cell types generated?
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Two different models to do this:
o Model one: one progenitor for each cell
type
o Model two: Population of progenitors that
are all similar, but are multipotent and can
generate all the different cell types of the
retina.
How to distinguish between these two different
possibilities?
o A question still asked for many parts of the
developing embryo, too, as we don’t
understand how the different cell types of
organs are generated. Do they generate from specific
Figure 5: Models of retinal cell
progenitor cells that make one cell type only, or, from a
multipotent progenitor cell?
o The retina serves as a model to understand these progenitor cell specifications.
o One way we could distinguish between these two models is by labeling the different cell
types…
Cell lineage studies – Label individual RPCs and analyze their progeny
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If there are 7 different progenitor cells each generating one cell
type, you would expect one cell only yielding progeny.
If you label a “grandparent” cell, you can follow whether it
differentiates into neural or glial cells.
Question: Are the differentiated daughter cells the same type or
do they differ and contain combinations of different cell types?
Figure 6: Cell lineage model
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Lecture 11
Using retroviral vectors for cell lineage tracing
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Tool to infect only dividing cells, or diving retinal cells.
A normal retrovirus would get into the genome and manipulate the host machinery of a cell. You
don’t want this virus to be able to spread. People have modified the retrovirus genome to make it
replication incompetent. It can still infect the cell, but once it proliferates, it cannot cause cell
lysis.
The retroviral vectors we use also integrate the host cell’s genome. When a virus enters the
cell, it manipulates the host machinery to transcribe their RNA with reverse transcriptase. Using
long term repeat sequences, the genome of the retrovirus will integrate into the genome of the
host cell. When the host cell divides, it will carry the genome of the retrovirus. If you can
manipulate the vector to carry a reporter gene (PLAP, GFP, etc), it will also be replicated. Then,
you can use histochemical procedures to indicate which gene has been infected.
For example, if you infect the progenitor cell with a retroviral vector and let the retina develop
for a couple of weeks (as long as it takes to generate the different cell types) and then get clones.
These clones come from a single progenitor cell, that was infected, divided and generated into
different cell types. Right away can see heterogeneity in the position of these cones, suggesting
the progenitor cell is multipotent and can generate different cell types. If one progenitor
generated one cell type, would only see one cell type in each cone (not the case).
These retroviral vectors must be diluted so we only infect a few cells so they will be sparsely
located in the retina (so when there is proliferation and differentiation into a retinal cell, we know
whether it is a clone). The chance of finding two cells next to each other that were both infected
is pretty rare.
Retinal progenitor cells are multipotent
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Using the same vector as above, if the cell was infected at an early stage (E14), large clones of
many different cell types are generated.
Using the same vector as above, if the cell was infected at a late stage (P2), small clones of fewer
cell types were generated.
This suggests that at the late stage, progenitor cells have fewer options and they are unable to
generate the early born fates. When it reaches the end of retinal genesis it has lost the potential to
generate certain cell types. The reason the clones are smaller at late stage is probably because the
retinal progenitor cells will be fully proliferated by P7 and should be ready to differentiate into
neuronal and glial cells. Too much proliferation of the retinal progenitor cells could cause
problems such as retinoblastoma, a hyperproliferation of retinal ptogenitor cells, which forms
into cancer.
General conclusions from these studies
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There are no unipotent (fate-restricted) retinal progenitor cells.
RPCs are multipotent (but young progenitors have more potential than later progenitors).
Late progenitors have lost the capacity to generate early born cells (ie there were no early born
cells in clones generated from progenitors labeled late)
Labeling cells undergoing DNA synthesis at various time reveals birth date
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At P0, noticed some cell types that were missing. For example, don’t get ganglion or horizontal
cells. One possibility is that the cells have lost potential to make new cells, as these cells are
made at earlier stages where progenitors are more competent to generate these cell types.
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Lecture 11
BrdU is a molecule which you can inject into an animal. This will be taken up by the blood
circulation rapidly and willl be distributed throughout whole animal.
o The “d” stands for deoxy, so it can be incorporated into DNA even though it is a uridine
analog.
o The bromine acts as the antibody target for localization.
Concept:
o Inject BrdU.
o Cells that are in S-phase will incorporate BrdU at a specific time point in retinal
development, say E10, then wait until P7, for example.
o Cells uptake the BrdU but after each division, cells become less and less BrdU positive.
These cells will soon]
o 34 mins
Cells uptake the BrdU. Everytime a cell divides it loses BrdU, as it has time to proliferate cells
become less and less BrdU positive. Instead of going through mitosis it differentiates into a
neuron and it will be labeled in BrdU if it is a terminal cell type.
Look at population of neurons that actually had BrdU staining in retina. Count, using BrdU
antibodies and cell-type specific markers to determine what type of neuron they are.
Different retinal cell types are born at specific times
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People have injected BrdU into different cell types
and looked at BrdU staining in the retina.
Using BrdU antibodies and cell-type specific
antibodies they determined which neurons were
developed when.
Hoziontal cells, ganglion cells, and cone cells
were induced at early stages. They retain BrdU
labeling and were strongly BrdU positively.
The other cells had diluted BrdU when injected at
early stages. But when injected late, bipolar cells,
Muller cells, glial cells, were heavily stained with
BrdU.
Figure 7: Retinal cell types
How do RPCs choose to become a particular cell type?
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They are multipotent, they generate different cell types at specific times, but how do they
generate a specific cell type? This is important because it can be implicated in stem cell therapy.
If could regenerate retinal cells from stem cells could use in replacement therapy. In addition,
many neurodegenerative diseases would benefit from stem cell therapy. Ultimately, we want to
replicate developmental events in stem cell lines.
There are two major factors that affect cell specification:
o Environmental factors
 RGCs are one of the first cell types to be generated. It is possible that, as the
retina matures, the cellular environment changes such that it is not conducive to
RGC production (via secretion of some material)
o Intrinsic factors
 Are retinal progenitor cells intrinsically biased to generate particular cell types at
one time?
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Lecture 11
Environmental factors
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There was an experiment done in chicks in which they took early progenitor cells at E4 (in mice
at E10) and allow them to dissociate into a little ball of cells and place a membrane around them
which allows the fusion of molecules, but which the cells cannot pass. On the other side of the
membrane, you put the conditioning cells of increasing agents (start with some at E10 all the way
to P0).
At E10 there are no RGCs made in retina, but by E13-E14 they are generated. If you increase the
age of the conditioning cells, will you decrease the amount of RGCs generated?
Use the BrdU thymidine analog to label the cells that are proliferating and then stain with an
RGC marker to determine which were actually generated in dish in presence of those conditions
cells
Experiments I:
o Control - if the test cells are E4 in the presence of E4 condition cells, 35% RGCs result.
o If do this same experiment but in the presence of older aged cells, much less RGCs are
produced. In the late environment, there is a signal that prevents the generation of RGCs.
What cell type secretes this signal? Is it the RGCs themselves?
Experiment II:
o Take an E14 chick (E0 in the mouse), kill all the RGCs, do this experiment again, then
recover RGC production to level of control.
o Suggests the RGCs that are produced and secreting factors to inhibit production of further
RGCs. There are active feedback mechanisms in the retina.
Intrinsic factors
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Though RGCs seem under control of environmental factors, these factors don’t tell them what
cell type to become.
Question: Are RPCs intrinsically biased to generate particular cell types at any one time?
Experiment I: can the early environment force late progenitors to make early born cells?
o Take a late progenitor cells from P0 cells and put in presence of an excess of older cells.
Now look to see if can transform that cell and start making early progenitor cells.
Experiment II: Can the late environment force early progenitors to make late-born cells?
o Same pattern with opposite conditions.
Results: There is a change in cell fate decision.
o This tells us that RPCs have a strong intrinsic control whether they were generated at an
early or late stage. Seem to be doing so by intrinsic program develops that is modulated
by intrinsic feedback decisions in the environment. No matter what environment you put
the cell in, it will continue to do what it is supposed to do, suggesting a strong intrinsic
program in these cells.
A variety of transcription factors (TFs) operate in RPCs to regulate cell fate decisions
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What could be this intrinsic genetic program? There have been many studies looking at the the
production of different cell types, with what TFs influence what cell type.
For example, Foxn4 is a transcription factor expressed at early stages.
Foxn4 is expressed at the right place and time to specify amacrine and horizontal cell fates
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Note, the function of a gene is demonstrated only if:
o The gene is expressed at certain place and time where cell is
o The gene needs to be sufficient for that cell type
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o The gene needs to be required for that cell type
Since Foxn4 is expressed at the time amacrine and horizontal cells are produced (and not at later
stages), it probably has an effect in early born fate cells.
Is Foxn4 required for their expression?
Foxn4 inactivation blocks amacrine and horizontal cell production
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First inactivate Foxn4 by doing a knock out in mouse, then using cell-type specific markers for
comparison. In both horizontal and amacrine cells, we observe a strong reduction in the
expression of these cells.
This suggests Foxn4 function is required for production of amacrine and horizontal cells.
Is Foxn4 sufficient for their expression?
Foxn4 overexpression increases production of amacrine cells
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One can use retroviral vectors to force the
overexpression of Foxn4 in RPCs to see if can
induce more horizontal and amacrine cell
production.
GFP was used todenote Foxn4 and cell-type
specific markers were used to know which cell
type we were looking at.
Results
o There was a strong reduction in rod
photoreceptor cells.
o Strong increase in production of
Syntaxin+ cells (marker for amacrine and
horizontal cells).
Suggests Foxn4 is sufficient for production of
amacrine and horizontal cell…
Figure 8: Foxn4 overexpression
Asymmetric cell division contributes to generate cell diversity
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Could there be other intrinsic components controlling diversity? One of the key processes that
contributes to cell diversity that is conserved is a process called asymmetric cell divison. You
must divide asymmetrically to generate diversity,
otherwise, cannot make two daughter cells
Two methods of asymmetric cell division:
o Extrinsic – stem-cell niche:
 The mother cell divides which makes
two identical daughter cells. One
daughter cell exposed to environmental
signals sister cell does not see and
takes on different cell fate.
 The division itself was symmetric but
influence from environment was
asymmetric.
Figure 9: Asymmetric cell division
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Lecture 11
o Intrinsic
 Protein or mRNA that is asymmetrically segregated on cortex of mother cell, and
is asymmetrically inherited by daughters. Ends up in one of the daughters and not
other one – influences fate.
 It is intrinsic because happening inside cell and division itself was asymmetric.
Coordination of cell division orientation with polarized localization of fate determinants
generates intrinsic asymmetric cell divisions
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Using the polarity axis, cell polarizes expression of cell fate
proteins such that it is asymmetrically inherited with
daughter cell.
You can imagine that if the spindle was horizontal, would
get symmetric inheritance of protein and thus symmetric
division.
Figure 10: Asymmetric cell division
Mode of cell division: symmetric vs asymmetric
Figure 11: Symmetric cell division
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Figure 12: Asymmetric cell division
There is something that influences fate of cell, such as a transcription factor or a protein.
When the spindle is horizontal, there is a symmetric division of a factor. Identical cells will be
generated, either:
o Two progenitor cells – exponential expansion of progenitor cells
o Two terminal cells – neurons
 This type of division allows huge production of neurons in a short time.
When the spindle is vertical, there is asymmetric division of a factor. There are three different
groups of cells that could be generated:
o Two progenitor cells that are different from each other.
 For example, one could be fated to divide once more and the others to divide three
moer times. These are theoretically possible but difficult to study as no good
markers for progenitor cells.
o “Stem cell mode of division”
 Division in which a cell divides to make copy of itself, so there is self-renewal but
at the same time there is production of neuron.
o Terminal asymmetric division
 Two different types of neurons produced, which increases the diversity of postmitotic cell produced.
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Lecture 11
PCs divide along different planes
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Is there oriented cell division in the retina? You can see horizontally and vertically dividing cells
through their chromosome orientation. Perhaps there is asymmetrical inheritance in vertical
divisions
How could we know if vertical vs horizontal division affects fate?
Orientation of RPC division correlates with cell fate decisions
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Using time lapse electron microscopy, you can take the retina out of the animal, then take a slice
of tissue and put it on a filter (which are used to grow organs, example for skin grafts).
You can take a retina at E13 and put on this filter and it will follow normal development,
generating all the different layers for study ex vivo.
Using a retroviral vector to induce GFP expression in RPCs. These cells will divide on the apical
surface, and every 10 minutes you take a picture then reconstruct a movie. See whether it is
horizontal or vertical division.
In 90% of the time, when cells divide horizontally, two daughter cells of the same type result.
In 80% of the time, when cells divide vertically, the daughter cells become different types.
Suggests that cell division orientation is a random process; there is a strong correlation between
orientation of division and fate of cell.
Are there any asymmetric fate determinants that would regulate this?
Asymmetric inheritance of cell-fate determinants contribute to generation of cell diversity
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Numb “numbs the effect of Notch” ie is an antagonist of Notch. Notch is important in cellular
differentiation, so a protein that would inhibit Notch would affect cell fate.
Numb is asymmetrically localized to the cortex of cells, which would divide asymmetrically if
the spindle was vertical.
What would asyemmetric inheritance of Numb do? Must inactivate it to study it’s function.
Clonal genetic inactivation of Numb
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Study cell autonomous function
Allow temporal-specific inactivation
Lineage analysis
To inactive Numb, must do it clonally, in a single
cell. This cell would divide and make two more
progenitors, or two neurons of same type, or two
neurons of different type.
Figure 13: Clonal genetic inactivation of Numb
Take advantage of the Cre-Lox system, in which
LoxP are sequences that are integrated into the genome of the mouse, flanking an important axon
in the Numb gene. Cre Recombinase expression is induced and removes the region between the
LoxP sites.
In this experiment, they took the retina out of an animal, put on filters, then infected them with
Cre-expressing viral vector that is also expressing GFP. Cre inactivates Numb and GFP tells us
which cell was infected.
Then, you allow the retina to develop and differentiate for 17 days, fix the tissue, and use celltype markers to determine whether division was symmetric or asymmetric.
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When do this, don’t know if inactivation of this gene could lead to some non cell-autonomous
effect. Only want to inactivate Numb in a single cell, otherwise could disrupt signaling of some
secreted factors, for example, if affected many cells.
Can do this early, late, or in middle of retinal development and can identify lineage of entire
infected cells.
Inactivation of Numb increases symmetric divisions and decreases asymmetric divisions
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If you inactivate Numb, you find a
dramatic increase in the proportion of
symmetric divisions in versus asymmetric
divisions.
There is an increase in
photoreceptor/photoreceptor daughter
cells (for example) and a decrease in
production of terminal asymmetric
products (many types).
Suggests Numb is required for those terminal divisions.
But is Numb sufficient?
Figure 14: inactivation of numb
Numb gain-of-function increases symmetric terminal divisions at the expense of
asymmetric terminal divisions
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Now overexpress Notch, and would expect opposite results but actually observed the same
results.
In both cases, you broke the asymmetry by inactivating Numb or by overexpressing it and thus,
lose assymetric Numb presence in the daughter cells.
A model of Numb function in terminal asymmetric cell divisions
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In a normal animal, Numb is asymmetrically inherited and that
goes on to activate Notch in only ONE of the daughter cells (while
the other cell has Numb and thus inactivates Notch).
Now, when there is over-expression of Numb, you force
symmetrical inheritance in both cells so they both inhibit Notch
and so both take on same fate.
The asymmetric presence of those proteins during division instructs
asymmetric division. It doesn’t instruct a specific cell type but
instructs asymmetry or symmetry.
Figure 15: Numb function in terminal asymmetric cell division
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