February 06 Developmental biology: imaginal discs 5 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 Adult structures? time

February 06 Developmental biology: imaginal discs 6 What does this mean? A-P compartment division is now stable (and with it engrailed expression) 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 disc.

February 06 Developmental biology: imaginal discs 7 Where do 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.

February 06 Developmental biology: imaginal discs 8 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

February 06 wingless and hedgehog/engrailed and intersection with: Developmental biology: imaginal discs 9 Step 1: what gene activities “control” where discs are going to form? wingless expression A P AP decapentaplegic expression dorsal-ventral restricted stripe dpp expresssion in dorsal ventral axis: dpp distalless expression

February 06 Developmental biology: imaginal discs 10 In red: expression of distalless gene: trancription factor; distalless mutant misses extremities! Imaginal discs provide two (and three-dimensional?) models for patterning of complex structures?

February 06 7

Developmental biology: imaginal discs 11 * mathematical models for patterning of complex structures (often based on regenerating model-systems) Polar co-ordinate model: - repair by intercalation - not-Cartesian (non-flat) - beginning and end specified - local cell interactions Proposed by French and Bryant.

February 06 Developmental biology: imaginal discs 12 Boundaries model (Meinhardt): - embryonic patterning Truth?Is out there! - at least three “compartment” - intersection of these determines reference point - morphogenetic activities pattern

February 06 Developmental biology: imaginal discs 13 Reality? 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 wingles s dpp

February 06 Developmental biology: imaginal discs 14 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: secreted and act as morphogen molecules. Their concentration drives cells into particular differentiation pathways.

February 06 Developmental biology: imaginal discs 15 ptc mutant cells Expression of Ci Loss of Hh pathway components leads to molecular defects; as in embryos. Now targets are not embryonic epithelial cells forming cuticle but cells forming bits of leg/wing etc…. Hedgehog patched signalling downstream

February 06 Developmental biology: imaginal discs 16 normal wingabnormal increased Hh signalling ab abnormal decreased Hh signalling Loss or gain of Hh signalling leads to external defects; same for Dpp and Wg.

February 06 Developmental biology: imaginal discs 17 “mature” leg disc Only anterior-posterior axis determined. What about dorsal-ventral? 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).

February 06 Developmental biology: imaginal discs 18 Mathematical models and reality? - both models approach reality - non-cartesian model evokes compartments - morphogens proposed by Meinhardt. - both say distal point is high point and circular rings of cell determination descending

February 06 Developmental biology: imaginal discs 19 Hedgehog and wingless and signalling pathways and medicine? Cells induced to express oncogenic Smoothened Assay these cells for activation of pathway (increase of Ci transcription factor) All genes acting in pathway in segment, act here as well; onogenic smoothened leads to skin cancer

February 06 Developmental biology: imaginal discs 20 Dorsal-ventral patterning (molecular): “mature” wing disc Compartments like in anterior-posterior patterning: fringe (with Notch, Delta and Serrate) anterior-posterior compartment boundary gene dorsal-ventral compartment boundary gene Activity of these genes creates signalling centre at D/V boundary/.

February 06 Developmental biology: imaginal discs 21 “mature” leg disc Intersection of dpp/wingless and hedgehog: all signalling molecules at highest levels. How do you keep the “high” point in correct place? Hedgehog “non” signalling: inhibits expression of hh: via repressor factor (Gli in vertebrates) ( Active signalling ) And cell autonomous inhibition… More molecular mechanisms: Cells do NOT mix…. (mechanism not entirely understood) Plus:

February 06 Developmental biology: imaginal discs 22 “mature” disc Is this a model for patterning processes of fields of cells in other organisms? A patterning activity present in posterior cells? Seems to work as morphogen in anterior-posterior axis? Development of vertebrate limb: naïve group of cells

February 06 Developmental biology: imaginal discs 23 Search for morphogenetic activity in ZPA? Sonic hedgehog expressed in ZPA cells. Similar experiment in flies using human shh gene…. Ectopic placement of Sonic hedgehog protein in anterior:

February 06 Developmental biology: imaginal discs 24 Hh/Shh and signalling pathway molecules (segment polarity genes) drive anterior-posterior patterning in both fly as well as vertebrate limb * sonic hedgehog gene is expressed in group of posterior cells * sonic hedgehog activity drives anterior-posterior patterning in vertebrate limb (and in fly limb) * coincidence that it is the same genes? Effect of mutation in transcription factor downstream of hh (Ci=Gli) (gene identified in flies)

February 06 Developmental biology: imaginal discs 25 Sonic hedgehog expression in ZPA is responsible for anterior-posterior patterning in vertebrate limb. Hedgehog (sonic hedgehog) /wingless (Wnt) and dpp (BMP) signalling cooperate to set up 3-D paterning system (many genes originally found and characterized in flies) Many gene products (targets as well as interacting) connected to signalling in fly limb: conserved……...

February 06 Developmental biology: imaginal discs 26 Patterning of the simple discs in flies: - driven by patterning genes: secreted signalling molecules -wingless, hedgehog, dpp in flies: Wnt, hedgehog and BMPs in vertebrates - 3-dimensional structure building achieved by “simple” interaction between these molecules: high point of activity (generating most distal point) is high point of concentrations….. A fly is a human? A limb is a limb??????