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

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

3. chickalligator duckquail From www.saviorfare.wa & B.S. Arnold et al., 2001www.saviorfare.wa We have developed culture and imaging techniques to analyze avian development

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

5. Up to 5 days of imaging Embryo in natural setting Neural crest (from origin to destination) Up to 1 day of imaging Upright or inverted imaging Video and confocal time-lapse microscopy Intravital Imaging of Chick Embryos Whole Embryo Explant In ovo

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 Cells emigrate from all rhombomeresPrevious model hypotheses 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 Cells emigrate from all rhombomeresPrevious model hypotheses 1) Diffusion – Cells diffuse from specific segments (rhombomeres) (Le Douarin, 1995) PK & S. Fraser Dev. Biol., 1998 r3 r5 but avoid some areas

10. Highlights of Cranial Neural Crest Cell Patterning Cells can reroute their migratory pathsPrevious model hypotheses 2) Genetic – Cells are endowed with migration/destination instructions (Lumsden et al., 1991) 1) Diffusion – Cells diffuse from specific segments (rhombomeres) (Le Douarin, 1995) Premigratory neural crest cells ablated in r5-r6 wt 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 guidanceproliferation + N(x,y,t) ? ? ? ? ? ? ?

12. Highlights of Cranial Neural Crest Cell Patterning Our working model Rate of change in neural crest cells =chemotaxis+contact guidanceproliferation+ N(x,y,t) (cells proliferate during migration) (some cells follow one another after contact)

13. Highlights of Cranial Neural Crest Cell Patterning Our working model contact guidanceproliferation+ (cells follow one another, but can become leaders) ? ? ? ? ? Rate of change in neural crest cells =chemotaxis+ 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 guidanceproliferation+ 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 guidanceproliferation + N(x,y,t) ? ? ? ? ? Long range chemoattractant Areas of inhibition (cell-contact mediated)

16. Highlights of Cranial Neural Crest Cell Patterning Our working model Rate of change in neural crest cells =chemotaxis+contact guidanceproliferation+ (N(x,y,t)) Rate of change in chemical attractant =diffusion+productiondegradation+ (C(x,y,t)) Source of cells (midline) t = 0 L(t) 0 < x < L(t) Boundary moving at speed = s1 (um/hr) L(t) = L(0) +s1*t Long range chemoattractant at destination site

17. Highlights of Cranial Neural Crest Cell Patterning Our working model Rate of change in neural crest cells =chemotaxis+contact guidanceproliferation+ (N(x,y,t)) Rate of change in chemical attractant = (C(x,y,t)) Source of cells (midline) t = 0 L(t) 0 < x < L(t) Boundary moving at speed = s1 (um/hr) L(t) = L(0) +s1*t f (Diffusion, degradation, production,s1) Long range chemoattarctant at destination site Assume that C may be ~netrin (long range chemoattractant evidence from axon guidance studies

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

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 3) A cell links with another cell after contacting posterior end (evidence from dictyostelium (cAMP))

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 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? (evidence from dictyostelium (cAMP))

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 DiI Gfp via electroporation

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 DiI Projection of 30 um confocal sections Direction of motion

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