Lecture 18 Flashcards
What is the results of ectopic hedgehog signalling in the Drosophila appendage structures
Ectopic hedgehog signalling creates a mirror image duplication of limb structures
What are the Drosophila imaginal discs
Columnar epithelial cells of the ectoderm that give rise to adult structures
Other than limbs/appendages, what others adult structures are the imaginal discs for in the Drosophila embryo
Mouthparts, eyes/antennae, genitals
Imaginal discs are set up early in embryogenesis, T or F
T
Which segment of the Drosophila embryo contains the haltere imaginal disc
(Thoracic) T3
Which segment of the Drosophila embryo contains the leg imaginal disc
(Thoracic) T3
Which segment of the Drosophila embryo contains the wing imaginal disc
(Thoracic) T2
What is meant by a haltere
The haltere is a paired structure of the adult fly that is essentially its second pair of wings. However, over time, this structure has evolved a new role as a balance organ that counteracts the movement of the wings
The wing imaginal disc grows massively throughout development, increasing from 20/30 cells to 50,000+ cells. What is surprising about the structure during this period
The structure remains surprisingly similar throughout
There are three regions of the wing imaginal disc, what three adult structures do these regions give rise to
Notum, proximal wing and hinge, and the wing blade
What is mean by the wing imaginal disc developing akin to how an extendable aerial extends
The three concentric regions of the wing imaginal disc extend outwards forming the proximal-distal axis. The dorso-ventral axis is defined by a line the passes horizontally through the disc
The muscles that move the adult fly wing are found in the hinge, T or F
F – the wings themselves are stiff with all movements controlled by the muscles of the notum. The twisting motion of these muscles is translated by the hinge into the up and down action of the wing blade
How many cells thick is the adult fruit fly wing
2 cells thick
Bands of expression of which genes dictate the anterior-posterior and dorso-ventral axes
Dorso-ventral is defined by a band of wingless expression whilst anterior-posterior boundary is indicated by a band of ptc expression
Which axis forms the edge of the wing
Dorso-ventral
What three key genes are involved in dividing the anterior-posterior wing imaginal disc
Engrailed, hedgehog and decapentaplegic
What type of gene is engrailed
Engrailed is a segment polarity gene encoding a transcription factor
What is the effect on gene expression of engrailed
Engrailed switches on hedgehog which in turn induces wingless expression
Wingless and hedgehog work together to inhibit the formation of denticles, T or F
T
In early embryogenesis engrailed in involved in polarising segments, which region of the segment is indicated by engrailed expression
Engrailed defines the posterior compartment of each segment
What is similar about engrailed expression in the body segments and wing imaginal discs
Like the body segments, the wing imaginal discs also express engrailed only in the posterior. The presumptive imaginal discs inherit their anterior-posterior identity from the segmentation machinery.
Engrailed expression in the posterior segments pre-dates the regions where the imaginal discs form, T or F
T
Engrailed expression in the posterior wing, thoracic and abdominal segments is switch off in the adult fly, T or F
F - Engrailed expression remains on in the adult fly and it remains expressed in the posterior wing, thoracic and abdominal segments
What experimental evidence is there that engrailed is expressed in the posterior thoracic and abdominal segments as well as posterior wing
Creating a reporter construct encoding a engrailed-LacZ fusion protein drives expression of ?-galactosidase under the control of the engrailed promoter. Introducing the LacZ substrate X-galactose results in the production of a blue precipitate in the posterior segments etc.
What phenomena accounts for the distinct boundaries between engrailed expressing cells and those not expressing it
Cells expressing engrailed only having affinity for other cells expressing engrailed and vice versa. This is due to differences in intercellular adhesion
What is the result in hedgehog signalling in mutants with loss-of-function mutation in smo
Smo mutants lack hedgehog target gene repression and thus increased expression of target genes even in the absence of a hedgehog ligand
What is the effect of engrailed on Ci
Engrailed suppression Ci expression
What is the effect of engrailed on hedgehog expression
Engrailed induces hedgehog expression
What is the effect of engrailed on wingless
Engrailed upregulates expression of wingless. However, this isn’t direct and in fact, acts through hedgehog which upregulates wingless
Is Ci is only expressed in the absence on engrailed, where in the wing imaginal disc is it expressed
In the anterior compartment
As well as being the hedgehog receptor, Ptc is also a hedgehog signalling pathway gene upregulated by ligand binding, T or F
T
What can be said about the ptc expression pattern in the wing imaginal disc
Ptc is expressed in a band along the dorso-ventral axis
If ptc is a target gene of the hedgehog signalling pathway, then which cells is its expression confined to
Ptc can only be expressed in cells where hedgehog is present and Ci is not being repressed by engrailed
As hedgehog is only expressed in response to engrailed, its transcription is confined to the posterior wing imaginal disc, T or F
T
Explain using what you know about hedgehog why an expression band of ptc only occurs in the midline and initial anterior cells of the wing imaginal disc
As hedgehog is a morphogen it is made in the cells of the posterior wing imaginal disc and then is secreted. Hedgehog ligands then diffuse anteriorly to regions of cells that don’t express engrailed and thus haven’t had their Ci expression switched off. This means that the only cells capable of responding to the hedgehog induced in the posterior wing disc are those lying anterior of the midline where Ci is still being expressed. These cells will thus express ptc as a result of hedgehog target gene expression.
Decapentaplegic is another gene product involved in patterning the Drosophila wing. What causes dpp expression
Dpp is a hedgehog pathway target gene expressed as a result of hedgehog ligand binding
What type of patterning molecule is dpp
Dpp is a morphogen
Dpp is an invertebrate gene, what is its vertebrate homologue
TGF-?
Dpp is expressed exclusively in the midline of the wing imaginal disc, explain how this occurs
As dpp is a transcriptional target of hedgehog expression it can only be expressed in cells receiving hedgehog from the posterior wing disc cells where engrailed is being expressed. However these cells themselves need to not be expressing engrailed so that Ci expression isn’t inhibited. These cells occurs at the midline and slightly anteriorly and begin to express dpp. However, as dpp is a morphogen it diffuses both anteriorly and posteriorly creating a uniform decreasing gradient either side of the midline
Remembering that dpp is a morphogen and a homologue of the vertebrate TGF-?, what are the specific Type I and Type II receptors which it binds to
Tkv and Pnt
What invertebrate homologues of Smad proteins are activated as a results of dpp binding to type I and II receptors
Mad proteins
Brinker, omb and sal are target genes of dpp signalling, what are the specific effect of decapentaplegic on these genes
Dpp inhibits brinker expression and upregulates omb and sal genes
How can dpp’s action as a morphogen be visualised experimentally
Raising an antibody specific for phosphorylated mad proteins
Why can dpp protein be detected at a distance, using immunohistochemistry, in cells where in situ hybridisation experiments revealed no mRNA for that gene
Dpp is a morphogen
Experimental evidence for dpp’s effects on sal and omb expression revealed a much wider band of omb throughout the imaginal disc whereas sal expression was refined to a thinner band closer to the midline. Explain these experimental results
Sal requires a much higher concentration of dpp to be expressed by the cells. Hence sal isn’t expressed more distant from the midline because the concentration of dpp isn’t sufficient. In contrast there is sufficient dpp to induce omb expression throughout
What is meant by a morphotrap
A morphotrap is a membrane-tethered anti-GFP antibody that is a single chain antibody made by cammelids
In wild type embryo wing imaginal discs, where are the highest levels of brinker (brk) expression seen and why
The highest brk expression is seen at the periphery where ddp expression is at its lowest levels – dpp negatively regulates brk
What would be the effect of creating a morphotrap for GFP if you expressed it in cells under the control of the dpp promoter that also have a GFP motif knocked into the GFP gene
As morphotraps are membrane-tethered they will restrict the expressed dpp-GFP fusion protein to the cell surface effectively making it paracrine.
What would be seen experimentally if you restricted the diffusion of dpp away from the midline
A more refined band on dpp expression in the midline and brk expression projecting much more medially and forming a clear boundary rather than a gradient
Explain what would happen as a result of cloning cells in the posterior wing imaginal disc that have engrailed knocked out
These cells will express ptc due to a loss of repression of hedgehog target genes. The cells will be produce Ci because it isn’t being repressed by engrailed which has been knocked out. Hedgehog produced by neighbouring cells will bind to ptc receptors leading to expression of hedgehog target genes. These cloned cells will therefore express dpp because hedgehog is present and Ci is expressed
Explain what would happen as a result of cloning cells in the anterior wing imaginal disc that have forced engrailed expression
This will result in a border of dpp expression around the cells with forced engrailed expression. This is because Ci expression in those cells will be repressed by engrailed. Also engrailed expression causes hedgehog expression in those cells. This hedgehog will diffuse out of the cells where engrailed is expressed to the surrounding cells of the anterior wing imaginal disc. These anterior cells are wild type and do not express engrailed and thus are expressing Ci and can respond to hedgehog signalling. This will result in the activation of hedgehog signalling and expression of dpp
Explain what would happen as a result of cloning cells in the anterior wing imaginal disc that have patched knocked out
This will have the same effect as ectopic hedgehog signalling and will lead to dpp expression in those cells with ptc -/-. This is because ptc -/- will have decrease inhibition of smo. This means smo will be constitutively active even with the ligand absent. As Ci is also present because there is no engrailed anteriorly, dpp is expressed as well as other hedgehog target genes
What would be the result of a Ptc and Ci double knockout experiment in a region of cells in the midline, where dpp is normally expressed
Ptc and Ci double knockout in the midline will result in a further hedgehog diffusion anteriorly from those cells in the posterior where it is expressed. This is because hedgehog is not binding to any ptc receptors in the midline and so more is available to diffuse further.
What type of signalling protein is wingless
A morphogen
Wingless expression drives expression of pathway target genes in a concentration dependent manner and defines the dorsal ventral boundary in the same way that engrailed does the anterior-posterior axis, T or F
T
