1. Imaginal discs and what they teach us about morphogens petros.ligoxygakis@bioch.ox.ac.uk

2. wingless hedgehog/engrailed * after several hours of interdependence of wingless and hedgehog: situation changes. Development is a timed process; once a situation is established. Next one is set up… * engrailed expression is permanent in posterior compartment cells time Wingless/engrailed

3. Where do adult fly structures come from? Imaginal discs metamorphose into adult structures in pupal stage Each part of body grows and develops independently Each part of the adult fly is derived from an imaginal (imago=adult) disc.

4. Where do imaginal (derived from the imago=adult) discs come from? Each disc is derived from set of ectodermal cells in early embryo. During metamorphosis The flat sheets of cells, shoot out to from 3-D structure.

5. wingless and hedgehog/engrailed and intersection with: Step 1: what gene activities “control” where discs are going to form? wingless expression A P decapentaplegic expression dorsal-ventral restricted stripe in dorsal ventral axis: dpp distalless expression

6. Step 2: molecules that set up the patterning within disc segment posterior dorsal ventral hedgehog engrailed Disc derived from intersection of wingless/hedgehog and dpp expression (these activate distalless). “ mature ” disc Expression of these genes is maintained and drives patterning wingless dpp

7. Expression of dpp and wingless is maintained by hedgehog signalling Stable boundary from embryos (compartment boundary): forms the source of morphogenetic molecules. “ mature ” leg disc Patterning in anterior-posterior axis: Hedgehog, Wingless, decapentaplegic Their signalling drives cells into particular differentiation pathways.

8. 17 “ mature ” leg disc Intersection of dpp/wingless: In a body part sticking out: proximal-distal axis…? Intersection between dorsal-ventral and anterior-posterior defines proximal-distal axis! High point is most distal! In fact, dorsal ventral boundary was already there in embryos (cells see either green or pink or combinations of these). Distalless expression defines limb extremities

9. What is a morphogen I: The “classic” definition by Lewis Wolpert A morphogen is a diffusible molecule that elicits direct long-range concentration-dependent changes in gene expression and (in the end) in cellular behaviour. Seductive idea because: it provides a simple correlation between specific substances (morphogens), an input (their concentration) and an output (the response of the cells, which is expressed as a developmental pattern).

10. What is a morphogen II The key attributes of morphogens — on the basis of the classical definition and on present perceptions — are: They are secreted, diffusible molecules that come to be distributed in a concentration gradient from a fixed spatial source. They generate several (at least three) discrete cellular states in response to different thresholds of the concentration gradient. These cell states are usually associated with differential gene expression. They are instructive (that is, they function as a determinant of the cell state) in a direct manner (that is, without intermediates; the response of the cells does not depend on the cell changing states first). Examples of molecules and situations that fulfil the criteria above are: Spatzle, specifying the dorsoventral axis of the Drosophila melanogaster embryo. Bicoid, specifying the anterior-posterior axis of the Drosophila embryo Activins, specifying and patterning the mesoderm during the early development of amphibians and other vertebrates. Sonic Hedgehog, in vertebrate limb development

11. 22 A patterning activity present in posterior cells Works as morphogen in anterior-posterior axis Development of vertebrate limb: naïve group of cells What is a morphogen III: Sonic Hedgehog in vertebrates

12. wingless hedgehog/engrailed Uniform overexpression of wingless: modest expansion of engrailed time Wingless as a “classic” case of a morphogen (or?) time Set up by pair rule wingless hedgehog/engrailed Ubiquitous wingless Engrailed is expressed only in competent cells

13. In pupae, discs turn into structures (by extension mainly), cells have a “ molecular definition ”. * Can we use development of fly wings/legs as a model for “ making ” an extremity (understand the underlying genes)? * Study molecular basis of disc patterning…. Fate map * Study morphogens….

14. Zecca M, Basler K & Struhl G Direct and Long-Range Action of a Wingless Morphogen Gradient, Cell, Volume 87, Issue 5, 1996, 833 - 844

15. UAS FRT CD2, y + FRT Wg tag FLP UAS CD2, y + Wg tag GAL4 Clone induction at 1 st instar Observation at 3 rd instar THE FLP-out technique

16. Zecca M, Basler K & Struhl G Direct and Long-Range Action of a Wingless Morphogen Gradient, Cell, Volume 87, Issue 5, 1996, 833 - 844

17. C Alexandre et al. Nature 505, 180-185 (2014) doi:10.1038/nature12879 Characterization of membrane-tethered Wingless expressed from the wingless locus.

18. Gene expression in wingless-null mutant patches surrounded by wild-type or Neurotactin–Wingless-expressing cells. C Alexandre et al. Nature 505, 180-185 (2014) doi:10.1038/nature12879

19. Activity of the wingless promoter in the prospective wing. C Alexandre et al. Nature 505, 180-185 (2014) doi:10.1038/nature12879

20. Tissue-specific allele switching to assess the contribution of Wingless release to organ- autonomous growth rate and organismal developmental timing. C Alexandre et al. Nature 505, 180-185 (2014) doi:10.1038/nature12879

21. No long range function for Wingless Since Wg does not act at a distance then it is not a morphogen with the classical sense of the definition. An idea that held gospel status in developmental biology. Time to revisit Wnts? Tear up the textbooks? Or as a famous victorian lady commented on Darwinism: “Let’s hope that it is not true but, if it is true let us make sure that it is not widely known”