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Paul Kulesa Stowers Institute for Medical Research

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.

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Paul Kulesa Stowers Institute for Medical Research

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  1. Insights into vertebrate development: merging bioimaging and computational modeling Paul Kulesa Stowers Institute for Medical Research

  2. 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

  4. Intravital Imaging of Chick Embryos Whole Embryo Explant • Up to 1 day of imaging • Upright or inverted imaging • Video and confocal time-lapse microscopy

  5. 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)

  6. 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)

  7. 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?

  8. 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

  9. 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 PK & S. Fraser Dev. Biol., 1998 but avoid some areas

  10. Highlights of Cranial Neural Crest Cell Patterning Previous model hypotheses Cells can reroute their migratory paths 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

  11. 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) ? ? ? ? ? ? ?

  12. 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)

  13. 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 = chemotaxis + contact guidance + proliferation N(x,y,t) ? ? ? ? ? Lu, Fraser, & PK, Dev Dyn. 2003

  14. 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

  15. 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 ? ? ? ? ?

  16. 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 < x < L(t) t = 0 Source of cells (midline) Long range chemoattractant at destination site L(t) = L(0) +s1*t

  17. 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 < x < L(t) t = 0 Source of cells (midline) Long range chemoattarctant at destination site L(t) = L(0) +s1*t

  18. 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

  19. 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

  20. 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

  21. 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?

  22. 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

  23. 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

  24. 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

  25. r4 r5

  26. 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

  27. 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

  28. Somites form slightly slower in whole embryo culture

  29. Gfp labeled blood cells in early circulation GFP transgenic mouse line from M. Baron/Mt. Sinai

  30. It is important to maintain the embryo in one place P. Trainor

  31. Acknowledgements • Stowers Institute for Medical Research • Paul Trainor • Caltech • Scott Fraser • Marianne Bronner-Fraser • Mary Dickinson • Dave Crotty

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