1 time * transcription factors expressed in large blocks Developmental biology: segment polarity genes and cell signalling b i c o d g a p e n s time * transcription factors expressed in large blocks * induce expression of downstream transcription factors in smaller blocks * generates segmental expression (14) of segment polarity genes. February 06 1

2 Non-cellular: syncitium Developmental biology: segment polarity genes and cell signalling Non-cellular: syncitium Transcription factors are able to determine more than just the nuclei that produce them February 06 2

3 How do you relate the molecular expression of Developmental biology: segment polarity genes and cell signalling How do you relate the molecular expression of segment polarity genes in stripes in cells in every segment, to the final larval structure….? February 06 3

4 Each segment is affected in a wingless mutant: Developmental biology: segment polarity genes and cell signalling normal wingless mutant Each segment is affected in a wingless mutant: cells making “naked” cuticle are mis-patterned and are lost (death) or redirected to form denticles February 06 4

5 * mutants: each segment polarity mutant shows Developmental biology: segment polarity genes and cell signalling * mutants: each segment polarity mutant shows identical defect repeated in every segment * conclusion: segment polarity proteins are required for pattern formation in each and every segment * different classes: role? February 06 5

6 * detailed analysis of expression. Developmental biology: segment polarity genes and cell signalling * detailed analysis of expression. * wingless expressed in one cell wide stripe February 06 6

7 * wingless gene encodes for a secreted signalling protein; Developmental biology: segment polarity genes and cell signalling expression of wingless gene * wingless gene encodes for a secreted signalling protein; Int-1 (now Wnt-1) gene cloned in mice as being involved in breast cancer * Secreted molecules being involved in patterning consistent with fact that embryo is now cellularised! February 06 7

Developmental biology: segment polarity genes and cell signalling * how do segment polarity genes generate pattern? Targets of signalling? engrailed expression in normal embryo engrailed expression in wingless mutant embryo wingless engrailed * wingless acts on neighbouring cells to maintain engrailed expression. February 06 8

9 * hedgehog gene encodes for a secreted signalling protein. Developmental biology: segment polarity genes and cell signalling * wingless acts on neighbouring cells to maintain engrailed expression. * hedgehog gene encodes for a secreted signalling protein. wingless hedgehog * wingless also acts to maintain expression of hedgehog gene in neighbouring cells * target of hedgehog signalling? February 06 9

Developmental biology: segment polarity genes and cell signalling wingless expression in normal embryo wingless expression in hedgehog mutant embryo wingless hedgehog * two signalling molecules maintain each others expression: maintain a stable reference point in segment February 06 10

11 hedgehog wingless ? How does the “boundary” get there…. Developmental biology: segment polarity genes and cell signalling hedgehog wingless ? How does the “boundary” get there…. Pair rule gene activity Anterior precise’sh (posterior not); Segment polarity interplay refines expression domains and sets up segment boundaries (later, see textbooks). February 06 11

Developmental biology: segment polarity genes and cell signalling Stability? Field of cells: virgin area where no/little prepatterning has taken place. What is the most efficient way of turning this into something useful for the animal? * simple (as little genes/protein as possible) * reliable * accident resistant February 06 12

secreted signal: efficient!? Damage: ie loss of blue cell Developmental biology: segment polarity genes and cell signalling Damage: ie loss of blue cell is devastating…. growth secreted signal: efficient!? Blue cell has to be fixed to stay blue Not all daughters can stay blue February 06 13

identity is actively kept Developmental biology: segment polarity genes and cell signalling * blue and green cell identity is actively kept growth * loss of one blue cell is corrected * regeneration…. February 06 14

* regeneration…. 15 flatworms amphibians Developmental biology: segment polarity genes and cell signalling amphibians * regeneration…. flatworms Molecules that drive regeneration important for stem cell technology February 06 15

16 * genetic studies to unravel segment patterning: Developmental biology: segment polarity genes and cell signalling * genetic studies to unravel segment patterning: wingless hedgehog identical phenotype: molecules work to common end wingless expression in smo-/- Smo Hedgehog protein X February 06 16

17 * patched mutant phenotype is very different from Developmental biology: segment polarity genes and cell signalling patched normal * patched mutant phenotype is very different from hedgehog or wingless mutant phenotype wingless hedgehog * what does this mean “molecularly”? February 06 17

18 * overexpression of hedgehog using a transgene, leads to same Developmental biology: segment polarity genes and cell signalling normal patched mutant overexpression of hedgehog gene * overexpression of hedgehog using a transgene, leads to same phenotype as patched mutant patched = hedgehog everywhere patched mutant February 06 18

19 patched-hedgehog double mutant patched mutant = Developmental biology: segment polarity genes and cell signalling patched-hedgehog double mutant patched mutant = hedgehog over-expression hedgehog patched hedgehog normal patched mutant * patched hedgehog double mutant: looks like patched single mutant. hedgehog patched signalling downstream February 06 19

20 patched-hedgehog double mutant Developmental biology: segment polarity genes and cell signalling patched-hedgehog double mutant * patched gene expression in all cells that do not express hedgehog gene. * patched required downstream of hedgehog: inhibits signalling hedgehog patched signalling downstream * patched gene encodes for twelve transmembrane protein. * patched protein can bind hedgehog protein February 06 20

21 hedgehog Smo Ptc Fu Su(fu) Cos2 Ci Ci Developmental biology: segment polarity genes and cell signalling Ptc hedgehog Smo Cos2 Fu Su(fu) Ci Ci February 06 21

26 hedgehog protein processing: proteolytic activity Developmental biology: segment polarity genes and cell signalling hedgehog protein processing: proteolytic activity cholesterol addition acyltransferase * patched protein contains sterol sensing domain February 06 26

22 http://www.stanford.edu/~rnusse/wntwindow.html Developmental biology: segment polarity genes and cell signalling February 06 22 http://www.stanford.edu/~rnusse/wntwindow.html

23 * two secreted signals (hedgehog and wingless) that Developmental biology: segment polarity genes and cell signalling * two secreted signals (hedgehog and wingless) that are required to pattern each segment. * other segment polarity genes encode for proteins that act either in hedgehog or wingless pathway. wingless hedgehog * fine patterning of segment: late roles for wingless and hedgehog. February 06 23

24 * patterning via signalling proteins is most common way of Developmental biology: segment polarity genes and cell signalling wingless hedgehog * patterning via signalling proteins is most common way of development (cellular mode of development). * signalling processes conserved? YES! * wingless is fly homologue of a vertebrate oncogene: Wnt-1 * patterning molecules are heading a differentiation/growth decision pathway! February 06 24

29 Roles for segment polarity proteins in other animals? Developmental biology: segment polarity genes and cell signalling Roles for segment polarity proteins in other animals? in development: midline (ventral Hedgehog; dorsal wnt) In patterning of arms/legs In cell differentiation (neurons, germline cells, “organs”) In control of cell growth (skin, mammary gland) Ageing? February 06 29