Recruitment of cells into the - PowerPoint Presentation

1. Recruitment of cells into the Drosophila wing primordium by a feed-forward circuit of vestigial autoregulationMyriam Zecca and Gary Struhl

2. Important questionsWhat is a feed-forward mechanism?What are Drosophila wing primordium and wing imaginal disc?What are vestigial (vg), apterus (ap) and Wingless (Wg)?What is DSL (Delta/Serrate/LAG-2)-Notch?

3. Feed-forwardIt’s a term describing an element within a control system that passes a controlling signal from a source in its external environment, to a load elsewhere in its external environment (opposite of feedback).

4. What are the Drosophila wing primordium and the wing imaginal disc?The Drosophila wing primordium is a group of cells that will form the wing later on in development. It is defined in a discrete subpopulation of cells within the wing imaginal disc Population of epithelial cells that will give rise to cuticular and ephitelial structures of the adult body.It’s divided along the proximal-distal axis into regions that will form the body wall, also called notum (proximal), wing hinge (central) and wing blade (distal) in the adult.

5. Adult wings are formed by two epidermal layers (dorsal & ventral surfaces) held together. The imaginal discs undergo extensive morphological changes to produce a wing. Mechanisms and genes responsible are very similar to those of the vertebrate limb.

6. What are vestigial (vg) and Wingless (Wg)? What is DSL (Delta/Serrate/LAG-2)-Notch?vestigial (vg) is a ‘selector’ gene that specifies the wing. Selector genes control cell and tissue behavior by regulating downstream ‘realisator’ genes (Garcia-Bellido, 1975).Selector genes present two kinds of behavior: inherited expression vs active signaling:MDDActivated by inheritanceCsignalCActive signalingmitosis

7. Wingless (Wg):It’s a long-range morphogen that drives vg expression in surrounding cells up to 25-30 cell diameter away.Morphogen: a substance whose non-uniform distribution governs the pattern of tissue development in the process of morphogenesis or pattern formation.DSL-Notch signaling: It’s a short-range signal that in this case induces D-V border cells to express vg and to secrete Wg.apterus (ap)It’s a gene activated in the dorsal (D) compartment by inheritance, and segregates the disc into dorsal (D) and ventral (V) compartments.

8. How do they all work together in the imaginal disc?The vg gene is first activated in a small cluster of cells, defining the wing imaginal disc, and is later on expressed in a central portion of the disc until it’s segregated into dorsal (D) and ventral (V) compartments by the heritable activation of ap in D compartment cells. Following the D-V segregation, cells in both compartments are programmed to activate DSL-Notch signaling across the D-V compartment boundary inducing vg expression in a thin stripe of ‘border’ cells flanking the boundary, and also inducing them to secrete Wg.Decapentaplegic (Dpp), secreted by A compartment cells along the A-P compartment boundary, also upregulates vg away from the D-V compartment boundary, centering an expanding population of vg-expressing cells on the intersection between the boundaries.AP1°2°and vg3°

9. … so what’s the problem here?Wg and Dpp signaling are found together in many different contexts during Drosophila development, yet they only induce cells to express vg in the wing disc. Hence, it appears that Wg and Dpp can only recruit cells to express vg if they are already defined as prospective wing, a state that should itself depend on pre-existing Vg activity.This paradoxical requirement suggests a crucial, but as yet unresolved, role for vg in controlling its own expression in response to Wg and Dpp.

10. What’s the novelty in this work?They provide evidence that Wg promotes the rapid expansion of the wing primordium following the D-V segregation by fueling a non-autonomous circuit of vg autoregulation.They show that vg-expressing cells send a short-range feed-forward signal that is required to entrain neighboring cells to upregulate vg in response to Wg. They also show that this process can reiterate from one cell to the next, propagating the recruitment of surrounding cells into the wing primordium.

11. How did they do this?They focused on how Wg signaling controls vg expression and wing growth by taking advantage of ap mutant discs.In ap mutant discs, border cells are not specified, the early expression of vg that normally precedes the D-V segregation dissipates, and the wing primordium fails to develop. By generating clones of cells that ectopically express Vg, Wg or both, they showed that cells within ap mutant discs could be recruited to express vg in response to Wg, but only if they were located near or next to cells that already express Vg.

12. More important conceptsEctopic expression: expression of a gen in cells that wouldn’t express it in normal conditions.Autonomous trait: only genotypically mutant cells exhibit the mutant phenotype.Non-autonomus trait: genotypically mutant cells cause other cells (regardlles of their genotype) to exhibit a mutant phenotype.Exogenous DNA: DNA that originates outside of the organism of study.Endogenous DNA: DNA that originates inside the cells of the organism of study.The GAL4-UAS system is a biochemical method used to study gene expression and function. The system has two parts: the Gal4 gene, encoding the yeast transcription activator protein Gal4, and the UAS (Upstream Activation Sequence), an enhancer to which GAL4 specifically binds to activate gene transcription.

13. ResultsControl of vg expression by the boundary and quadrant enhancers (Fig. 1A-D)The ‘border’ and ‘pouch’ expression domains of vg are mediated by distinct boundary and quadrant enhancers (BE and QE). They monitored the activities of these enhancers by assaying transgenes that express reporter proteins under their control.Fig. 1

14. Wg signaling is necessary but not sufficient to activate the quadrant enhancer Supplying ectopic Wg to ap0 discs failed to rescue the expression of vg, QE reporter genes, or other ‘pouch genes’(Fig 1G-H).Hence, Wg is not sufficient to activate QE-dependent vg expression or to rescue wing development in the absence of D-V border cells.Non-specificstainingThe pouch in ap0 discsis either absent or present just like a small cluster ofrn-only cellsFig 1

15. In agreement with the model, clones of cells that express Notch in ap0 discs behaved as ectopic border cells and rescued wing growth (even when generated up to 48 hours after the time at which D-V segregation would normally occur).Fig 2

16. Non-autonomous activation of the quadrant enhancer in response to ectopic Vg-expressing cells and Wg.They conclude that Vg-overexpressing cells have the capacity to induce neighboring cells to activate QE-dependent vg expression, but only if the responding cells also receive Wg.Moderate levels of ectopic Vg can act together with Wg to activate the quadrant enhancer in neighboring cells.Cells that express moderate levels of exogenous Vg, well within the normal physiological range, can drive QE-dependent vg expression in neighboring cells, provided that the responding cells also receive Wg signalSo what happens at normal physiological levels?

17. Distinct Wg- and Vg-dependent signals act combinatorially to recruit prospective wing disc cells to the wing fateRecapitulating: Wg can only induce wing disc cells to express high levels of vg and develop as wing cells only in the presence of cells that already express Vg.Possible explanation: Vg activity programs cells to make a second intercellular signal that is required in combination with Wg. Test: they analyzed ap0 wing discs that contained neighboring Tub1>vg and UAS-Nrt-wg clones (C-D). Tub1>vg clones were able to induce cells within neighboring UAS-Nrt-wg clones to express peak levels of both vg and the 1XQE reporter, provided that the two types of clones abutted.Important: this vg expression can spread through the Nrt-Wg-expressing clone and extend to adjacent cells outside the clone.Fig. 6

18. DiscussionTheir main finding is that Wg is not sufficient to sustain or induce vg expression ap0 discs. Instead, Wg can only drive vg expression in these discs when the responding cells are near or next to cells that express exogenous Vg.That led them to propose this model: DSL-Notch signaling (purple) induces wg (red) and BE-dependent Vg (VgB, green) expression in border cells, causing them to send Wg (red arrows), as well as the short-range feedforward signal, ‘X’ (black arrows). X entrains neighboring cells to activate QE-dependent Vg expression (VgQ, blue) in response to Wg, and these cells become a new source for X propagating recruitment of surrounding cells into the growing wing primordium.Fig. 8D-V boundaryborder cells

19. AcknowledgementsI would like to thank my partners from SysBio group for their constant support.I’m also very thankful to Luis that helped me to understand the present paper, and Carole who is not present and helped me to rehearse the presentation.