Regulation of Cell Proliferation by a Morphogen Gradient

1
Regulation of Cell Proliferation by a Morphogen
Gradient

• Dragana Rogulja and Kenneth D. Irvine
• Cell, Col. 123 449-461
• November 4, 2005
• Presented by
• Jessica Wang Sarah Chan

2
This paper is about

• Drosophila development
• Wing growth
• How morphogen gradients influence cell
  proliferation
• Cell responses
• Autonomous and non-autonomous

3
Imaginal Disc

• Patches of cells that will form various body
  parts (e.g., antennae, legs, wings).
• In the wing imaginal discs, genes such as
  engrailed, wingless, hedgehog, and
  decapentaplegic (DPP) are important for
  patterning

Wolpert et al. 2002
4
Decapentaplegic (DPP)

• Morphogen
• Important in Anterior-Posterior patterning in the
  wing
• Controls wing growth
• Overexpression of DPP (or its active receptor
  TKV) causes wing overgrowth

DPP
TKV (Thickveins)
MAD
Brinker
transcription
5
So

• We are talking of a morphogen gradient
• MORPHOGEN (Wolpert et al. 1998)
• substance active in pattern formation
• spatial concentration varies
• to which cells respond differently at different
  threshold concentrations

6
Recall
Different morphogen
Different response

Wolpert et al. 2002
7
To Clarify

• Previous example
• Cell response colour
• Determining factor threshold of morphogen
• In the Paper
• Cell response cell proliferation
• Determining factor relative positional value

8
Gradient models postulate

• Distinct positional values reflect cells
  location within gradient
• Cells compare position with their neighbors
• Differences in position stimulate cell divisions
• Daughter cells positional values intermediate
  between neighbors
• Discrepancies become more shallow until
  proliferation ceases

Model used to explain tissue regeneration in
insects and amphibians (French et al. 1976)
9
Expectations

• DPP gradient present in imaginal disc
• ? DPP ? growth
• ? DPP ? growth

DPP
BUT
(Day and Lawrence, 2000)
10
Wait! A couple of glitches
How do we get even growth?
DPP gradient
growth
?

• Uniform expression of DPP/activated TKV causes
  wing overgrowth
• Is the level of DPP rather than the slope
  important?

11
Methods

• Drosophila larvae Mosaic and Wild-Type
• Mosaics
• exhibits mutant and wild-type phenotype
• have transgene AyGal4PR
• Used transgene for temporal control
• Visualized effects of transgene on wing growth
  using stains

12
The making of the Transgene

(Struhl and Basler, 1993)
Biol 430 Dr. Ian Ching-Sang
Flp-out cassette
Gal4-UAS system

13
AyGal4PR
Transgenic larvae RU486 food
0 h
5 h
But no transcription of gene
10 h
14
A Technicolor Experiment Stains

• GFP (Green) shows us where TKV is being
  expressed
• BrdU (red) shows us where DNA is replicating
  (I.e., growth and proliferation)

15
Two types of Proliferation

• Autonomous
• Only cells that have the mutant genotype show TKV
  induced cell proliferation
• Non-Autonomous
• Mutant cells cause wild-type cells to show
  TKV-induced cell proliferation

16
Autonomous Effects I

• Increase in cell proliferation in clones located
  in lateral regions
• Wild-type levels of DPP/activated TKV are lowest
  in the lateral regions

17
Autonomous Effects II

• Clones in medial regions showed little autonomous
  cell proliferation

18
Autonomous Effects III

• Clones in medial-lateral regions showed
  intermediate effects depending on their position
  on the disc

19
Nonautonomous Induction of Cell proliferation by
activated TKV in medial cells
Lateral ? non-autonomous
Medial ? non-autonomous
20
Summary

• Autonomous response
• Uniform TKV activity
• ?Lateral growth
• ?Medial growth
• Non-Autonomous response
• Difference in TKV activity between cells
• Lateral growth unchanged
• ? Medial growth
• Results suggest
• In medial cells, relative TKV activity important
  for cell proliferation

21
Testing the Hypothesis I
Non-autonomous proliferation is induced by
juxtaposition of cells with different positional
values

• Uniformly activate TKV
• Only cells in lateral regions show proliferation
• Proliferation in medial regions is inhibited
• Therefore, medial proliferation requires a TKV
  gradient

22
Testing the Hypothesis II
Non-autonomous proliferation is induced by
juxtaposition of cells with different positional
values

• Perhaps the level of TKV is too high?
• Lower the level of TKV in cells (from 20µg/ml to
  1µg/ml)
• Proliferation is weaker, but the pattern is the
  same
• Therefore proliferation depends on difference in
  TKV levels between neighbour cells

proliferation
23
Testing the Hypothesis III
Non-autonomous proliferation is induced by
juxtaposition of cells with different positional
values

• If proliferation depends on differences (not
  pathway itself) we should be able to get the same
  effects by inhibiting the DPP pathway
• GFP attached to a DPP pathway inhibitor
• Cell proliferation greatest at clone borders,
  where greatest difference in levels is

No DPP activity
24
The Punchline

• Medial cell proliferation depends on slope of
  gradient NOT absolute TKV activation (where there
  is no slope)
• uniform TKV inhibits cell proliferation in medial
  wing
• Recall Lateral growth autonomous response
• Suggests level of TKV important NOT gradient

25
Distinct strategies for growth regulation

• Two distinct mechanisms by which DPP pathway
  regulates growth
• 1) Depends on gradient of DPP (medial)
• Nonautonomous
• 2) Depends on level of DPP (lateral)
• Autonomous

26
Autonomous Gradient responses to TKV activity
27
Gradient response equal throughout, but gradient
shallow in lateral, steep in medial
Autonomous response low in medial wing, high in
lateral wing
28
Steep DPP gradient
Shallow DPP gradient
Autonomous gradient response Gradient
response Autonomous response
Overall equal growth
High in medial
High in lateral
29
Almost NO gradient
Overall, lateral overgrowth
Medial no response Lateral growth spikes
Autonomous gradient response Gradient
response Autonomous response
No gradient, no response
30
Clones, cells with high TKV
Very steep gradients
Autonomous gradient response Gradient
response
Autonomous response
spikes in growth in response to extreme gradient


Not affected by gradients
31
But how do cells actually do this?
32
Mechanism
Cell membrane
Neighbour cell
TKV
Unbound X
x
Transmembrane protein
Bound X
Growth
Localizes X so X goes where it is unbound and is
removed from the side where it is bound
33
Mechanism II
Asymmetric distribution of X can be passed on
from cell to cell
Ma et al 2003
34
Mechanism II
Asymmetric distribution of X can be passed on
from cell to cell
growth
35
Mechanism II
Asymmetric distribution of X can be passed on
from cell to cell
Daughter cell
36
Final Thoughts I

• Prior failures due to
• Nonautonomous response transient
• Overgrowth of lateral regions had obscured
  inhibition of medial-cell proliferation
  associated with uniform TKV activation
• Unique methods and protocols used

37
Final Thoughts II

• Dual regulation mechanisms could extend to other
  parts of the organism (e.g., other discs, or
  other areas regulated by DPP)
• Candidates for X can be identified and
  elucidated is it specific to DPP?
• Cytonemes (Hsiung et al., 2005) a possible role
  in DPP signaling as presented in this paper?

Your final thoughts Questions?
38
References

• Day, S.J., and P.A. Lawrence. 2000. Measuring
  dimensions the regulation of size and shape.
  Development 127 1257-1264
• French E.V., Bryant, P.J., and S.V. Bryant.
  1976. Pattern regulation in epimorphic fields.
  Science 193 969-981
• Hsiung, F., Ramirez Weber, F.A., Iwaki, D.D. and
  T.B. Kornberg. 2005. Dependence of drosophila
  wing imaginal disc cytonemes on Decapentaplegic.
  Nature 437 560-563
• Ma, D., Tang, C., McNeill, H., Simon, M.A. and
  J.D. Axelrod. 2003. Fidelity in planar cell
  polarity signalling. Nature 421 543-547.
• Struhl, G., and K. Basler. 1993. Organizing
  activity of wingless protein in drosophila. Cell
  72 527-540