Paul Kulesa

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Insights into vertebrate development merging
bioimaging and computational modeling
Paul Kulesa Stowers Institute for Medical Research
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Insights into vertebrate development merging
bioimaging and computational modeling
Paul Kulesa Stowers Institute for Medical Research
3
We have developed culture and imaging techniques
to analyze avian development
chick
alligator
duck
quail
From www.saviorfare.wa B.S. Arnold et al., 2001
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Intravital Imaging of Chick Embryos
Whole Embryo Explant

• Up to 1 day of imaging
• Upright or inverted imaging
• Video and confocal time-lapse microscopy

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Intravital Imaging of Chick Embryos
Whole Embryo Explant

• Up to 1 day of imaging
• Upright or inverted imaging
• Video and confocal time-lapse microscopy

In ovo

• Up to 5 days of imaging
• Embryo in natural setting
• Neural crest (from origin to destination)

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Craniofacial Patterning Cell migration and
guidance
Model system The Neural Crest
Cutis, 1999

• Incorrect migration can lead to birth defects
• Frontonasal dysplasia
• Waardenburgs syndrome (pigment)
• Neurofibromas (peripheral nerve tumors)

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Craniofacial Patterning Cell migration and
guidance
Model system The Neural Crest
Cutis, 1999

• Incorrect migration can lead to birth defects
• Frontonasal dysplasia
• Waardenburgs syndrome (pigment)
• Neurofibromas (peripheral nerve tumors)

How do cells sort into and maintain migrating
streams?
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Highlights of Cranial Neural Crest Cell Patterning
Cells emigrate from all rhombomeres
Previous model hypotheses
1) Diffusion Cells diffuse from specific
segments (rhombomeres) (Le Douarin, 1995)
PK S. Fraser Dev. Biol., 1998
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Highlights of Cranial Neural Crest Cell Patterning
Cells emigrate from all rhombomeres
Previous model hypotheses
1) Diffusion Cells diffuse from specific
segments (rhombomeres) (Le Douarin, 1995)
r3
r5
PK S. Fraser Dev. Biol., 1998
but avoid some areas
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Highlights of Cranial Neural Crest Cell Patterning
Cells can reroute their migratory paths
Previous model hypotheses
1) Diffusion Cells diffuse from specific
segments (rhombomeres) (Le Douarin, 1995)
wt
2) Genetic Cells are endowed with
migration/destination instructions (Lumsden et
al., 1991)
Premigratory neural crest cells ablated in r5-r6
Cell trajectories are disrupted
PK, Bronner-Fraser, S. Fraser, Dev., 2000
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Highlights of Cranial Neural Crest Cell Patterning
Our working model
Rate of change in neural crest cells

chemotaxis
contact guidance
proliferation


N(x,y,t)
?
?
?
?
?
?
?
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Highlights of Cranial Neural Crest Cell Patterning
Our working model
(some cells follow one another after contact)
Rate of change in neural crest cells

chemotaxis

contact guidance
proliferation

N(x,y,t)
(cells proliferate during migration)
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Highlights of Cranial Neural Crest Cell Patterning
Our working model
(cells follow one another, but can become leaders)
Rate of change in neural crest cells
contact guidance
proliferation


chemotaxis

N(x,y,t)
?
?
?
?
?
Lu, Fraser, PK, Dev Dyn. 2003
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Highlights of Cranial Neural Crest Cell Patterning
Our working model
Rate of change in neural crest cells

chemotaxis

contact guidance
proliferation

N(x,y,t)
Average cell speed 49 - 9 um/h Average
directionality 0.29 - 0.1

• Cells at the stream fronts
• higher directionality (28)
• slower avg speed
• directed filopodia

Cell tracking w/J. Solomon S. Speicher/Caltech
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Highlights of Cranial Neural Crest Cell Patterning
Our working model
Rate of change in neural crest cells

chemotaxis
contact guidance
proliferation


N(x,y,t)
Areas of inhibition (cell-contact mediated)
Long range chemoattractant
?
?
?
?
?
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Highlights of Cranial Neural Crest Cell Patterning
Our working model
Rate of change in neural crest cells

chemotaxis

contact guidance
proliferation

(N(x,y,t))
Rate of change in chemical attractant

diffusion

production
degradation

(C(x,y,t))
L(t)
Boundary moving at speed s1 (um/hr)
0 lt x lt L(t)
t 0
Source of cells
(midline)
Long range chemoattractant at destination site
L(t) L(0) s1t
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Highlights of Cranial Neural Crest Cell Patterning
Our working model
Rate of change in neural crest cells

chemotaxis

contact guidance
proliferation

(N(x,y,t))
Assume that C may be netrin (long range
chemoattractant evidence from axon guidance
studies
Rate of change in chemical attractant
f (Diffusion, degradation, production,s1)

(C(x,y,t))
L(t)
Boundary moving at speed s1 (um/hr)
0 lt x lt L(t)
t 0
Source of cells
(midline)
Long range chemoattarctant at destination site
L(t) L(0) s1t
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Highlights of Cranial Neural Crest Cell Patterning
Our working model
(some cells are repelled from an area after
contact)
Rate of change in neural crest cells

chemotaxis

contact guidance
proliferation

N(x,y,t)
(some cells are attracted to other cells to form
a chain like array)
r6
r7
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Highlights of Cranial Neural Crest Cell Patterning
Our working model
(some cells are repelled from an area after
contact)
Rate of change in neural crest cells

chemotaxis

contact guidance
proliferation

N(x,y,t)
(some cells are attracted to other cells to form
a chain like array)

• Highlights of chains
• Neural crest chains are made up of 5-10 cells
• May be a general mechanism of cell migration
• Chains form in neuronal precursors migrating to
  the
• olfactory bulb (Alvarez-Buylla, 2002)
• Tumor cells form chains in 3D collagen gels
• (Friedl, 2002)
• Dictyostelium (slime mold) form chains to
  assemble
• a multicellular organism

r6
r7
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Highlights of Cranial Neural Crest Cell Patterning
Our working model hypotheses (Discrete model for
contact guidance term)
1) Cells in the chain are linked together by
filopodia
2) A cell within a chain emits a chemoattractant
at its posterior end
(evidence from dictyostelium (cAMP))
3) A cell links with another cell after
contacting posterior end
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Highlights of Cranial Neural Crest Cell Patterning
Our working model hypotheses (Discrete model for
contact guidance term)
1) Cells in the chain are linked together by
filopodia
2) A cell within a chain emits a chemoattractant
at its posterior end
(evidence from dictyostelium (cAMP))
3) A cell links with another cell after
contacting posterior end
Main assumption for all 3 hypotheses Either lead
cell chews a hole in the extracellular matrix
(ECM) or ECM is permissive and lead cell lays
down a trail for others to follow.

• Simple model (cellular automata)
• Define a lead cell
• Lead cell moves mostly in lateral direction
• Leaves open spaces behind which other cells may
  move into
• Gives clues as to how close the lead cell must
  stay to attract followers
• Can leave behind clues instead of open spaces,
  such as chemoattractant
• short or long range interactions?

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Cellular structure of the chains
Our working model hypotheses (Discrete model for
contact guidance term)
1) Cells in the chain are linked together by
filopodia
DiI
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Cellular structure of the chains
Our working model hypotheses (Discrete model for
contact guidance term)
1) Cells in the chain are linked together by
filopodia
Gfp via electroporation
DiI
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Cellular structure of the chains
Our working model hypotheses (Discrete model for
contact guidance term)
1) Cells in the chain are linked together by
filopodia
Direction of motion
Projection of 30 um confocal sections
DiI
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r4
r5
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Do cranial neural crest cells in mouse migrate
with a rich set of behaviors?

• Challenges
• 3D embryo
• Gas exchange important
• Finer temperature control
• than in chick

• Benefits to Mouse culture and imaging
• Genetics (target mutations of genes related to
  craniofacial patterning)
• Several mutant mouse models available with
  craniofacial defects

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Do cranial neural crest cells in mouse migrate
with a rich set of behaviors?

• Challenges
• 3D embryo
• Gas exchange important
• Finer temperature control
• than in chick

Jones et al., Genesis 2002
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Somites form slightly slower in whole embryo
culture
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Gfp labeled blood cells in early circulation
GFP transgenic mouse line from M. Baron/Mt. Sinai
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It is important to maintain the embryo in one
place
P. Trainor
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Acknowledgements

• Stowers Institute for Medical Research
• Paul Trainor

• Caltech
• Scott Fraser
• Marianne Bronner-Fraser
• Mary Dickinson
• Dave Crotty

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