Lecture 9 - Dorsal closure in Drosophila as a model for
12:31 PM 3, 2014
Using the basic knowledge we learned
through in vitro situ and models to
understand the complexity of these events.
CSB331 Page 1 • If you cut yourself (not a massive wound,
you can see the wound mesenchyme
which is the underlying tissue.
• Imagine the epidermis is cut. This is the
basement membrane. And this is under
the filtration of the immune system. This
is going to reseal and this is a contractile
event we are talking about
•A cut uncovers the mesenchyme, • This is a chick embryo they used
underlying tissue. • When we look at embryogenesis, one of
•Resealing the epidermis involves a the reasons we scar is because we have
an immune system.
•We scar because we have an immune • Now people sell bandages that sell
system. immune suppressors.
•In pre natal development you can heal
• In pre natal development you can still
without scarring, because the immune heal without scarring. But once your
system is not yet developed.
immune system kicks into a normal
•Once immune system kicks into place, level there is scarring. This is an early
you get scarring. chick embryo so no scarring.
Keratinocytes move along and myosin motor protein is concentrated in the actin
• The word mesenchyme word applies to
fibroblast like environment in early
embryogenesis where the cells are on
their way to the final differentiation
• This is wound closure and a SEM. This
looks like a dough wrapped with a
• Below the PM there is the actin cables
where the cells are held together by TM
like receptors like e cadherin.
• It almost look like the actin myosin cable
is extracellular that’s because the
• Now you want to close this wound
SEM of a similar would reveals the superficial layer of epithelial
cells ( the periderm) as the sheet moves forward (upwards) to
cover the wound mesenchyme
fibroblast is a type of cell that synthesizes the extracellular matrix and collagen, the structural framework (stroma) for animal tissues,
and plays a critical role in wound healing. Fibroblasts are the most common cells ofconnective tissue in animals.
Mesenchyme is a type of tissue characterized by loosely associated cells that lack polarity and are surrounded by a large extracellular
matrix. Mesenchymal cells are able to develop into the tissues of thelymphatic and circulatory systems, as well as connective
tissues throughout the body, such as bone and cartilage. A sarcoma is a cancer of mesenchymal cells.
CSB331 Page 2 If you stain for components such as anti
myosin, non muscle myosin, you can see it
concentrated in the leading edge of the
epithelia. You are using propidium iodide
which stains in DNA in fluorescent red.
(the Daphne? Stains in blue). You can see
the leading edge of the E cells in the nuclei
and eventually it is going to close the
Fruit fly is taken as a model to look at
wound closure, affect signaling events
Left to right called dorsal closure and the involvement
of Rho factors involved in it. Rac, cd42 and
You inhibit the inhibitors of rho A you are
going to stop this. Because you need Rho A
to activate ROCK and effectors
Anti- myosin antibodies! --> acto myosin cable
CSB331 Page 3 • A female can lay a lot of eggs
• A fruit fly is used over a 100 years to use
as a model for genetic crosses.
• A large % of the molecules that make up
the FF are actually structurally and
functionally served in us.
• Highlights the point that you can use
model organisms to answer questions
that are not answerable in humans.
• One of the most used genetic tools is
the GAL4/UAS systems
• Imagine I have a protein and I want to express Rho
A or actin.
• I can control the expression of actin in a FF by
having an UAS element upstream of a gene
• This is a promoter element several tandems
upstream of a activator sequence and derived
• If I look for a UAS element in a fly genome I am
not going to find it
• The beauty of finding it in yeast is there is a
Reproduce 2 weeks and lots
of eggs so fast genetic specific protein that will bind to UAS and turn on
studies the expression of the gene
• Eg: I want to express GFP actin
If need to express a specific
gene have an activator ○ The gene G could be GFP actin
sequence and a TF upstream ○ If I can activate this sequence selectively, I can
turn on the expression of G actin.
• How do I control that it gets activated in a certain
This is not available in drosophila
type of cell and a certain stage in development?
Two constructs are inserted into the genome of the transgenic animal
1. Yeast specific regulatory sequence - UAS (Upstream 2. Coding sequence of the yeast Gal4 gene.
Activating sequence), coupled to a copy of the coding ○ Product is a yeast specific regulatory protein that
sequence of Gene g. see image. binds to UAS element.
○ Expressed: and controlled by the regulatory
region of gene H
• When GENE H is expressed --> Gal4 gene is ALSO
expressed --> Drives transcription of gene H.
CSB331 Page 4 • You can have a tissue specific regulator
sequence that is upstream of a GAL4
• Once GAL4 expression is turned on we
are ending up with the expression of
GAL4 which is an activator that loves to
bind to UAS element and turn on the
expression of the gene.
• This Is the way to selectively turn on the
expression of the gene, knowing that you
cannot find the Gal4 is not found in any
other promoter sequence.
• Then you engineer the fly to have a
specific regulatory sequence.
- Coding sequence for the Gal4 gene
• If I add the promoter for alpha beta actin
here, wherever in the normal fly the
promoter is going to be active, turn on
gal 4. and in this case I turn on GFP actin
where the normal actin is expressed.
• If I wanted to express selectivelyGal4 in
the hemocytes there is a peroxidase
promoter that will turn on GAL4 only
when the blood cells of the fruit fly and I
would express of the G actin only in
• This is called driver line
• There is 100s of these lines available
• I can have peroxidases and promoters
that gets activated that drives the
promotion of GAL4. I made that fly with
the G actin construct.
• Depending on the promoter you use, you
drive the expression of the gene in a
CSB331 Page 5 • Why the FF?
• Within 2 weeks you emerge adult flies
• You get early cleavages
Within 24 hours larvael
development • You get a syncytial blastoderm with
Only gastrulation stage cellularization
• Here is near the end of gastrulation, the
• There is larval stages, three instars,
pupae stage etc
• All these massive events have appeared
• And they are maturing with 24 hours
• There is the speed they grow
• They can survive on minimal material
○ Eg: apple juice
• There is no recombination in the male
so you can do complex things.
Dorsal Closure: takes place progressively,
during germ band retraction.
• It takes about two hours, beginning 11
hours after the start of development.
Dorsal closure is the process whereby
the stretched amnioserosa is covered by
epidermal cells that will ultimately fuse
at the dorsal midline. With its part in
development played, the amnioserosa is
then absorbed by the yolk. Genes
involved in dorsal closure include rho,
hemipterous and basket.
CSB331 Page 6 Germ band extraction --> pulls pole cells in --> withdraws and leaves an opening ( • You have to close the extra embryonic
space and that is dorsal closure.
• There is a germ band extension. There is
• Germ band extraction pulls the pole
cells right in.
• Then it withdraws and leaves an
• Even though the cells are not
highlighted, its called amnioserosa (The
amnioserosa is an epithelium that
derives from about 200 cells at the
dorsal midline of the blastoderm
embryo. It is required for proper germ
band extension and dorsal closure. ) and
then it is sealed like a wound.
• It is an absolute powerful system.
We need three entities"
- Extra embryonic epithelium cells - seal the gap Check this:
-Amnioserosa cells - fill the gap http://www.embl.de/aboutus/communic
- Actin band - Act like drawstrings to pull the cellation_outreach/media_relations/2009/0
Movement of the germ band to anterior
Eventually the extension is completed bringing germs cells to the termini
the germ band extension and retraction leaves the AS yolk rich tissue opened (like an opened wound)
Initially, the fertilized egg divides many times and becomes a hollow ball of cells (called cellular blastoderm). The posterior end of the
embryo (to the right) migrates up and over the top (dorsal surface) of the embryo in a process called germband extension, which is
responsible for delivery of specific cell types to the interior of the embryo. After this is accomplished, the reverse process (called
germband retraction) occurs leaving a large open hole on the dorsal surface of the embryo.
• Dorsal closure then takes place to close the hole by zipping up the surrounding epidermis over the amnioserosa cells, which overlays
the dorsal hole. At this point, the embryo has completed embryogenesis and is ready to proceed with hatching as a larvae
• Look how filo podia, lamellipodia and
actin cable all are sealed.
• You have dorsal closure, this gap is there
and the epithelium is going to seal it.
Then the amniserosa can be seen
• Rho is going to control it. You can
selectively knock out individual Rho
○ Deleing the gene
○ Creating a dominant negative form
with no GTPase activity
○ Or a form where it is always active
ventral • I can drive expression of Rho factors and
almost any cell type using the Gal4
• Another trick is if I would create a laser
wound, then I destroy the epithelia,
killing 2-3 cells are like 1-2cm in our
skin. If you wanna compare dorsal
closure to a wounding event, where
would you hit it with the laser?
I want to compare dorsal closure to a wounding event, where would you hit it with a laser?
• You are targeting the ventral side because if you hit the dorsal then you will be hitting the amnioserosa.
• I have a cell with a leading edge. I want to move it forward right at the leading edge. I want to give one fracture and a
receptor tyrosine kinase. A TM receptor with kinase activity. I have GF which is a chemoattractant.
CSB331 Page 7 receptor tyrosine kinase. A TM receptor with kinase activity. I have GF which is a chemoattractant.
• What happens is: we have phosphorylation (auto)--> PI3 kinase binds --> which recruits a GEF binding directly to it -->
activating Rac GTPase --> Rac. Rac stimulates PI3K --> WASP --> Arp complex gets activated --> gets rid of CAP proteins -->
• If I was going to throw in Rho, it has a likely chance of going to MLC. Separate them both in your minds.
• Have an understanding of generic molecules. What is the most common way to keep these molecules inactive? By folding.
By folding they are hiding cryptic hiding sites. Eg: when we pull talin, we exposed pip binding domains. We exposed
integrins biding sites. And other targets. Proline rich regions.
This type of sealing also occur in mammals
As a result of dorsal closure and the movement of lateral side the embryo is not too wide after
Epithelial cells are moving over the amnioserosa
• Tehre is a germ band extraction and the tail of the
emebryo comes forward and retract. During the
extraction process the amnioserosa becomes
exposed, the extraembryonic tissue. The idea is that
you are going to mvoe the opposing sheets of
epithelia to clsoe the amnioserosa close. The E. cells
are migrating on the cells that form the amnioserosa.
• Point: epithelial cells don’t move collectively. You can
have solitary cell movement, cluster of cell movement
and interaction of cell movement.
• You can use various staining methods. As we go
through the process here, you can see how dynamic
this is. You have suturing and finally closure.
• If you super activated Rho you get stress fibres. Super
activated Rac in GTP form --> you get membrane
rough link. CDC42 --> micro spike filopodia.
• Rac and cdc42 act in synchrony.
CSB331 Page 8 Immediately after the “initiation” phase,
leading edge cells begin to polarize as
filamentous actin accumulates at their
apical edge to form a thick actin cable. The
assembly of this cable coincides with, and is
probably responsible for, transformation of
the leading edge from a scalloped edge into
a taut, neat row of cells. Simultaneously,
the leading edge cells elongate in a
dorsoventral direction; it is not clear
whether they actively elongate or are
tugged forward by the purse-string activity
of the contractile actin cable at their “free
The zippering phase of dorsal closure begins
once opposing leading edges at the most
anterior and posterior ends of the hole are
close enough for filopodia to reach across
and touch the opposite epithelial edge
(Jacinto et al., 2000). These actin
protrusions act to zip the opposing
epithelial surfaces together and may also
play a key role in ensuring the correct
matching of the embryonic segments as the
hole closes. However, in addition to
filopodia, it is highly likely that both
contractility of the actin cable and
amnioserosa cell contraction still play roles
in bringing the sides together.
CSB331 Page 9 As for the other phases of dorsal closure,
the final stage of this process exhibits a
temporal progression, spreading like a wave
within the seams behind the two advancing
zipper fronts. A crucial aspect of
termination is that once leading edge cells
from opposing sides have made contact
they stop moving and form a tight seam
along the midline.
How does the epithelial cells close with a
CSB331 Page 10 • Amnioserosa cells are large
• Youa re staining this for alpha catening.
• If you make this transgene, what kind of promoter
will I use to drive the expression?
○ Gene x - a-catenin that is fused to GFP
○ Promoter will be - UAS
○ Turn the promoter on with Gal4
○ Tissue specific sequence drives Gal4.
• a-catenin as a protein that promotes adherence
• The image doesn’t tell you much about the
Can't tell where the
Actin promoter to express GAL4
And cross with a line that expresses GFP
to form a epithelial cell line in which GFP
is expressed temporally and spatially as
TEM - transmission electron micrsocope
• Mechanism gives you great insight into the dynamic state
• Fusion will suture themselves. 'at some point they fuse
and you have further differentiation of the cells.
CSB331 Page 11 canthus: the outer or inner corner of the
eye, where the upper and lower lids meet.
• Dorsal closure of the FF looks like the fusion in mammals.
• Some of the process of molecules involved between the
fruit fly of the mammals.
• At the mouse embryo too, you get cellular extenstion
at the leading edge, lamelipoda etc
• Some of the process in the mouse embryo is also
shared wbtween the ff and the mammals
CSB331 Page 12 • Wound healing:w e can look at dorsal closure.
• We can laser cells on the ventral side and watch the
• Idea: we are going to see if there is a division of labor
or prominent effect within the CD42 , Rho and Rac. If
we hit about 5 cells, you have essentially about 2-3
cm equivalent of wound.
• There are three different Racs that are targeted?
• Sometimes you have sub families of rac or rho you
have functional competitions.
Have filopodia Don’t have filopodia
• Absence of the cable and protrusions at the leading edge. You destroyed the system , and it is not fusing. Rac is
involved not only in the dorsal closure. You get the parallel effects in the ___.
• The two members of the rac faimly, rac 1 and rac2. if you do it with rac 1 you don’t see a severe phenotype.
• Rac 2 you see a severe phenotype.
CSB331 Page 13 The two model systems are comparable
• This is dorsal closure. Other is a laser wound on the
ventral side of the embryo.
• If you look at micrographs you see the same kind of