The Role of the JNK Signaling Cascade in Drosophila Imaginal Disc Eversion

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The Role of the JNK Signaling Cascade in
Drosophila Imaginal Disc Eversion

• Presented By Suzan Ergun

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Summary of Presentation

• Introduction
• Imaginal Discs
• Mechanisms of Eversion
• Paper
• Overview
• JNK Signaling
• Data Collected
• Conclusions
• Future Questions

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INTRODUCTION TO DISC EVERSION
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To Review
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Definitions

• Imaginal Discs
• Small sacs of epithelium present in the larvae of
  Drosophila and other insects, which at
  metamorphosis give rise to adult structures
  (Beddington et al., 2003)
• Monolayered epithelial invaginations
• Set during embryogenesis grow and invert during
  larval stages evert during metamorphosis
• Discs give rise to all adult structures except
  abdomen

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• Discs are composed of two distinct groups of
  cells
• Columnar cells
• Imaginal epithelium
• Adult structures
• Squamous cells
• Peripodial epithelium and stalk
• PS cells

http//biology.clc.uc.edu/courses/bio105/tissue.ht
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Adapted From (Pastor-Pareja et al., 2004)
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Imaginal Discs

• Imaginal discs undergo many morphological changes
  during metamorphosis
• Evert
• Expand
• Fuse to adjacent discs
• Disc eversion has been poorly studied

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• One mechanism predominant in the literature
  (Wolpert et al., 2002)
• Known role of molting hormone ecdysone
• The release of this hormone coincides with
  morphological transitions
• Sufficient to induce eversion in vitro (Milner,
  1977)
• Shown that contraction of the peripodial
  epithelium is necessary for eversion to occur
  (Milner, 1984)

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PAPER OVERVIEW

• Invasive Cell Behaviour During Drosophila
  Imaginal Disc Eversion Is Mediated by the JNK
  Signaling Cascade
• Jose Carlos Pastor-Pareja, Ferdinand Grawe, and
  Enrique Martin-Blanco

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This Paper

• 1) Presents a new morphological and cellular
  mechanism for disc eversion in Drosophila
• 2) Proposes the involvement of the JNK signaling
  cascade
• 3) Generates results using histological sections
  and direct observations in vivo

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Why JNK Signaling?

• In General JNK Signaling Pathway (Drosophila)
• Actin dynamics Lamellipodia and filopodia
  formation Cytoskeleton reorganization
  (Martin-Blanco et al., 2000)
• Regulates embryonic dorsal closure (reviewed in
  Martin-Blanco et al., 2000)
• D-Fos and D-Jun needed for disc eversion
  (reviewed in Pastor-Pareja et al., 2004)
• Controlled upstream by a
• signaling cascade similar to
• the JNK cascade in mammals

http//academic.brooklyn.cuny.edu/biology/bio4fv/p
age/cytoskeleton.html
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JNK Continued

• JNKK and JNK kinases key molecules in the
  mammalian JNK cascade
• JNK kinase homologous in Drosophila encoded by
  genes hemipetous (hep) and basket (bsk)
• JNK mutant larvae do not spread their discs
  phenotype accompanied by loss of puckered
  expression in the PS cells (Martin-Blanco et al.,
  2000)
• Puckered (puc) also involved in cascade
• Phosphates
• Inactivates bsk
• Role in negative feedback
• of JNK cascade

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Observations

• At the 3rd instar stage the disc and larval
  epidermis are separated by their extracellular
  basil lamina
• The initiation of pupation results in a loss of
  basil lamina between both surfaces
• Leads to adhesion of disc (peripodial side) and
  larval epidermis
• Therefore mechanisms looks to be more complicated
  than a simple contraction resulting from the
  release of ecdysone

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Observations

• At prepupa stage all PS cells (peripodial cells
  stalk cells) express puc
• Can use the pattern of puc expression to
  visualize PS cells during morphological changes

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Observations

• puc expression depends on JNK pathway
• puc expression nonexistent in hep larvae
• puc expression also nonexistent after a period of
  puc overexpression

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Observations

• During eversion only the cells found at the edge
  of the hole through which the disc everts and
  later at the leading front of the everting disc
  express puc
• Previously known that puc expressing cells do not
  change identity until adult structures form
• Topological dilemma histological samples show
  that these cells must reposition themselves
  inconsistent with old mechanism

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Observations

• The PS cells (puc expressing) have actin
  protrusions penetrating into the larval epidermis

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• Perforation of larval membrane by PS cell
  invasion was seen
• The larval epidermal surface shows expression of
  puc expressing PS cells
• Also the existence of several holes were seen in
  the PS/larval bilayer over time
• Within a few minutes these holes converge into a
  single hole

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Observations

• See progressive widening of hole by
  intercalculation of PS cells
• Retract and emit long filopodia and lamellipodia
  at front and rear ends of cells

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How Is This Possible?

• Cells need to physically relocate themselves
• But cells in tissue are connected
• Cell Adhesions
• Involve molecules that bind cells together and
  bind cells to the extracellular matrix
  (Beddington et al., 2003)
• Cells in insect tissue are connected by zonula
  adherens
• DE-cadherin, armadillo and Da-catenin
• Epithelial cells of flies also contain septate
  junctions

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Upon Close Examination

• All disc cells were found to contain ZA adhesions
• During disc eversion ZA components were found to
  delocalize from PS cell membranes and become
  cytoplasmic
• Only occurred in cells at the leading front of
  the disc and those at the edges of perforations
  in the PS/larval bilayer
• Septate junctions were also found to be displaced
• Cells became highly motile and displayed
  cytoskeletal reorganization

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• Therefore disc eversion requires a
  pseudo-epithelial-mesenchyme transition (PEMT) of
  PS cells
• Mesenchyme is tissue capable of migrating
  Invasive Cell Behaviour
• Process involves down regulation of cell-to-cell
  adhesions

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A New Mechanism

• Discs appose their peripodial membrane to the
  larval epidermis loss of basil lamina
• The larval epidermis is then invaded with PS
  cells (PEMT)
• The resulting PS/larval bilayer is perforated and
  hole is widened by intercalculation of cells
• When eversion is complete most PS cells head to
  the leading front of the disc and head disc
  expansion intercalculation of cells

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3 Classes of Defects

• Class I complete failure of PS/larval apposition
  (40)
• Class II disc apposes larval tissue but will not
  evert (50)
• Discs will evert completely and but will not fuse
  to other discs (10)

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Investigating the Role of the JNK Signaling
Cascade

• hepr75 mutants (hypomorphic) show range of
  defects (I-III)
• Complete inactivation of JNK by puc over
  expression before pupation results in severe
  defects 100 Class 1
• Delayed puc over expression results in less
  severe classes of mutations Class II III
• Conclude that the JNK is necessary for PS and
  larval cell apposition, eversion and fusion

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Investigating the Role of the JNK Signaling
Cascade

• JNK also found to effect the degree of PEMT in PS
  cells
• hepr75 mutants did not have delocalized ZA
  components in the cells at the leading front
• puce69 mutants (surplus of JNK activity) had
  enhanced cell motility and massive cell
  detachment from edges of epithelium
• Previous research indicates role in degree of
  motility

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In Summary

• JNK participates in
• puc expression in PS cells
• Apposition of PS and larval cells
• Regulation of adhesive properties of cells
  undergoing PEMT
• Maintenance of adhesion between larval and
  imaginal tissues

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Future Research

• Many areas still need to be investigated
• Some Questions
• How does JNK control PS cell shape and
  contraction?
• What is the role of ecdysone in all of this?
• What are the proteins involved in JNK activated
  PEMT?
• What are the steps leading to rearrangement of
  epithelial cells in fenestration?

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Literature Cited

• Cunliffe, L. 2004. Signalling a change. Nature
  Reviews 5, 867.
• Martin-Blanco, E., Pastor-Pareja, J., and A.
  Garcia-Bellido. 2000. JNK and decapentaplegic
  signaling control adhesiveness and cytoskeleton
  dynamics during thorax closure in Drosophila.
  Proceeding of the Natural Academy of Sciences
  97, 7888-7893.
• Milner, M. 1977. The eversion and differentiation
  of Drosophila melongaster leg and imaginal disc
  cultured in vitro with an optimal concentration
  of beta-ecdysone. Journal of Embryological
  Experimental Morphology 37, 105-117.
• Milner, M., Bleasby, A., and S. Kelly. 1984. The
  role of the peripodial membrane of leg and wing
  imaginal disks of Drosophila melongaster during
  evagination and differentiation in vitro.
  Developmental Biology 193, 180-186.