Lecture 14: drosophila appendage development

Question 1

Where are appendages arise from?

Answer 1

Many appendages are derived from imaginal discs which grow within the larva (quite passive) and become patterned. During metamorphosis the discs turn into the appendage structures.

Flies have ONE pair of wings, unlike most insects. Haltere = club shaped stub which flaps in opposite orientation to the way the wing beats. Allows flies to be very manoeuvrable and hover etc.

The physical signs of these segments can be seen in embryogenesis as gene expression. Thoracic segments 1-3 all have a leg disc. T2 has a marker of the wing and T3 has a marker of the haltere. The wing disc contains 50,000 columnar epithelial cells by the end of the third larval instar. Discs unfold into their adult shape through eversion where the imaginal disc edges come together.

Question 2

Explain patterning of the wing by morphogen gradients.

Answer 2

The D-V and A-P patterning of the wing disc sets up the structure allowing for wing formation. The axes are set up by morphogen signalling.

Wing imaginal discs ‘inherit’ their A-P identity from the segmentation machinery.

Engrailed (segment polarity TF) – expressed exclusively in and defines the posterior.

Engrailed expressing cells like to stick with other engrailed expressing cells and not with non-engrailed expressing cells. Therefore, as cells in the disc divide, they shuffle around to maximize contact with their cell type forming the straight line that defines the AP boundary.

Engrailed encodes Hedgehog (Hh) which is expressed in the posterior compartment.

Engrailed suppresses Ci, the downstream Hh TF preventing posterior cells from responding to their own Hh. Therefore, when engrailed is present there is no Ci and vice versa. Hh is a morphogen and diffuses into the anterior compartment of the wing disc. One of the target genes of Hh is Dpp (FGF-beta homologue).

In the anterior compartment there is no engrailed protein, and therefore the Wnt TF, Ci, can translocate to the nucleus and activate target genes. Dpp is therefore expressed along the AP boundary in the anterior compartment, and signals as a morphogen. Type 1 and type 2 Dpp receptors dimerise on activation, the TF is activatd (Mad) and activates omb and sal. At the highest concentrations of Dpp, closest to the source, omb and sal are expressed. Further away only omb is expressed.

The D-V axis of the wing disc is characterized by Wingless (wg) expression. Wg is a morphogen so diffuses away from the source. Apterous is expressed in the dorsal compartment.

A coordinate grip of A-P and D-V signals in the wing disc induces specific cell fates.

Question 3

Recall some classic experimental techniques supporting the process of wing patterning.

Answer 3

Transplanting HH (morphogen) into the posterior of the wing bud: diffuses to the anterior – sufficient to produce mirror image = symmetrical duplication of patterning.

Can dissect larva, stained with various markers to visualise what is happening in a gene expression level.

Enhancer trap line can stain cells.

Genetic screens.

Showing that engrailed defines the posterior compartment of the wing:

• en-lacZ reporter gene; lacZ expressed in place of Engrailed [protein] but under the control of the en enhancers (these enhancers are themselves controlled by the segmentation system)
• lacZ (b-galactosidase) converts soluble colourless X-gal into insoluble blue precipitate

Visualising the shape of dpp protein expression gradient is possible using phosphomad antibody staining (the TF activated in dpp signalling is Mad). It can also be detected from its expression using a dpp-lacZ enhancer trap domain. Protein moves away from zone of gene expression and can diffuse 4-50 cells away

Proving a molecule is a morphogen – stop it diffusing and see what happens:

• Using a morphotrap to hold Dpp-GFP on the membrane to disrupt gradient formation
• The expression domain is much tighter i.e., diffusion effected

Cloning

• Make a clone of cells mutant for engrailed in the posterior compartment
• No engrailed would mean Ci is no longer suppressed
• So downstream targets of HH made i.e., HH, Ptc and Dpp
• Dpp would diffuse into surrounding cells of the posterior compartment
• Made a clone of cells that express engrailed in the anterior compartment
• HH signalling out from clone
• Dpp diffuses out