Transcript Kulesa-Sloan-Swartz-July

Insights into vertebrate development:
merging bioimaging and computational modeling
Paul Kulesa
Stowers Institute for Medical Research
Insights into vertebrate development:
merging bioimaging and computational modeling
Paul Kulesa
Stowers Institute for Medical Research
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
Intravital Imaging of Chick Embryos
Whole Embryo Explant
• Up to 1 day of imaging
• Upright or inverted imaging
• Video and confocal time-lapse microscopy
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)
Craniofacial Patterning:
Cell migration and guidance
Model system: The Neural Crest
Cutis, 1999
Incorrect migration can lead to birth defects:
• Frontonasal dysplasia
• Waardenburg’s syndrome (pigment)
• Neurofibromas (peripheral nerve tumors)
Craniofacial Patterning:
Cell migration and guidance
Model system: The Neural Crest
Cutis, 1999
Incorrect migration can lead to birth defects:
• Frontonasal dysplasia
• Waardenburg’s syndrome (pigment)
• Neurofibromas (peripheral nerve tumors)
How do cells sort into and maintain migrating streams?
Highlights of Cranial Neural Crest Cell Patterning
Previous model hypotheses
Cells emigrate from all rhombomeres
1) Diffusion – Cells diffuse from
specific segments (rhombomeres)
(Le Douarin, 1995)
PK & S. Fraser Dev. Biol., 1998
Highlights of Cranial Neural Crest Cell Patterning
Previous model hypotheses
Cells emigrate from all rhombomeres
1) Diffusion – Cells diffuse from
specific segments (rhombomeres)
(Le Douarin, 1995)
r3
r5
but avoid some areas
PK & S. Fraser Dev. Biol., 1998
Highlights of Cranial Neural Crest Cell Patterning
Previous model hypotheses
1) Diffusion – Cells diffuse from
specific segments (rhombomeres)
(Le Douarin, 1995)
Cells can reroute their migratory paths
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
Highlights of Cranial Neural Crest Cell Patterning
Our working model
Rate of change in
neural crest cells
N(x,y,t)
=
chemotaxis + contact guidance + proliferation
?
?
?
?
?
?
?
Highlights of Cranial Neural Crest Cell Patterning
Our working model
Rate of change in
neural crest cells
N(x,y,t)
(some cells follow one another after contact)
=
chemotaxis + contact guidance + proliferation
(cells proliferate during migration)
Highlights of Cranial Neural Crest Cell Patterning
Our working model
Rate of change in
neural crest cells
N(x,y,t)
(cells follow one another, but can become leaders)
=
?
?
?
chemotaxis + contact guidance + proliferation
?
?
Lu, Fraser, & PK, Dev Dyn. 2003
Highlights of Cranial Neural Crest Cell Patterning
Our working model
Rate of change in
neural crest cells
N(x,y,t)
=
chemotaxis + contact guidance + proliferation
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
Highlights of Cranial Neural Crest Cell Patterning
Our working model
Rate of change in
neural crest cells
N(x,y,t)
=
chemotaxis + contact guidance + proliferation
Areas of inhibition
(cell-contact mediated)
?
?
?
?
?
Long range chemoattractant
Highlights of Cranial Neural Crest Cell Patterning
Our working model
Rate of change in
neural crest cells
(N(x,y,t))
Rate of change in
chemical attractant
(C(x,y,t))
=
=
chemotaxis + contact guidance + proliferation
diffusion
+
production
+
degradation
L(t)
0 < x < L(t)
Boundary moving at speed = s1 (um/hr)
t=0
Source
of
cells
(midline)
Long range chemoattractant
at destination site
L(t) = L(0) +s1*t
Highlights of Cranial Neural Crest Cell Patterning
Our working model
Rate of change in
neural crest cells
(N(x,y,t))
Rate of change in
chemical attractant
(C(x,y,t))
=
chemotaxis + contact guidance + proliferation
=
Assume that C may be ~netrin
(long range chemoattractant evidence from
axon guidance studies
f (Diffusion, degradation, production,s1)
L(t)
Boundary moving at speed = s1 (um/hr)
0 < x < L(t)
t=0
Source
of
cells
(midline)
Long range chemoattarctant
at destination site
L(t) = L(0) +s1*t
Highlights of Cranial Neural Crest Cell Patterning
Our working model
Rate of change in
neural crest cells
N(x,y,t)
(some cells are repelled from an area after contact)
=
chemotaxis + contact guidance + proliferation
(some cells are attracted to other cells to form a chain like array)
r6
r7
Highlights of Cranial Neural Crest Cell Patterning
Our working model
Rate of change in
neural crest cells
N(x,y,t)
(some cells are repelled from an area after contact)
=
chemotaxis + contact guidance + proliferation
(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
r6
r7
• 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
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
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?
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
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
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
r4
r5
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
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
Somites form slightly slower in whole embryo culture
Gfp labeled blood cells in early circulation
GFP transgenic mouse line from M. Baron/Mt. Sinai
It is important to maintain the embryo in one place
P. Trainor
Acknowledgements
Caltech
• Scott Fraser
• Marianne Bronner-Fraser
• Mary Dickinson
• Dave Crotty
Stowers Institute for Medical Research
• Paul Trainor