Lecture 9 - Dorsal closure in Drosophila as a model for wound healing.pdf

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Cell and Systems Biology
Maurice Ringuette

Lecture 9 - Dorsal closure in Drosophila as a model for wound healing 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 contractile event 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 stage. • This is wound closure and a SEM. This looks like a dough wrapped with a leading edge. • 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 resolution. • 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,[1] 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 surface. 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 Rho. You inhibit the inhibitors of rho A you are going to stop this. Because you need Rho A to activate ROCK and effectors downstream. 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 from yeast • 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 gene. • 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. Two inserts - UAS - 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 those cells • 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 specific model. 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 dorsal closure • 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 • Cheap • 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 ventral. • Germ band extraction pulls the pole cells right in. • Then it withdraws and leaves an opening. • 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 90626_Heidelberg/index.html 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. dorsal Then the amniserosa can be seen • Rho is going to control it. You can selectively knock out individual Rho either by: ○ 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 system. • 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 --> thermal fluctuations • 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 closure 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 edge”. 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 invisible suture 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 junctions. • The image doesn’t tell you much about the mechanism. Can't tell where the suturing occurs 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 Actin TEM - transmission electron micrsocope • Mechanism gives you great insight into the dynamic state of it. • 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 healing event • 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. H 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 cellular dynamics
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