Vinay S Mahajan Aarthi Chandrasekaran

1. Proposed role of paracrine loops for the mechanism of threshold generation in the community effect observed in Xenopus myogenesis Vinay S Mahajan Aarthi Chandrasekaran

2. Mesoderm induction in cap cells of Xenopus

3. Demonstration of Community Effect in Xenopus myogenesis Developmental Biology, Wolpert

4. eFGF ? Cell–cell signalling OR Diffusible community factor 2001, Gurdon et al. Community factorsare distinct fromMorphogens

5. How does a diffusing community factor help in sensing community size ? Monk’s Hypothesis: Community factor: diffuses out synthesized at constant rate CANNOT SATISFACTORILY EXPLAIN THRESHOLD GENERATION [eFGF] profile at radius=Ri [eFGF] at r=0 X X X X X X R c %Ri R

6. Autocrine loop eFGF eFGF gene eFGFR eFGFR gene Xbra gene Driver gene

7. Autocrine AND Paracrine effects “CYCLES”

8. Assumptions Cells differentiate in response to the eFGF concentration only after steady state is reached Number of receptors per cell (RT) is constant Diffusivity of eFGF is constant in the sphere eFGF is produced at a constant basal rate PL and degraded by first order kinetics Signal for autocatalysis assumed to be linearly related to [Rb] by an autocatalysis rate constant (KS). Endocytosed receptor-ligand complexes don’t signal.

9. ] [ dL dL 2 d2L 0 PL KS2 [Rb]2/4 α[L] - + + = + Deff [RT] [L] dt dr2 r dr [Rb] = KD + [L] dL = 0; r = 0 dr dL + P[L] = 0; r = R D dr MODEL EQUATIONS ] [ dL dL 2 d2L 0 PL KS1[Rb] α[L] - + + = + Deff dt dr2 r dr Boundary Conditions Rf + L Rb [RT] = [Rb] + [Rf] KS2 =4*KS1/ [RT] D Deff = “Ligand capture” D=Diffusivity [RT]KD P=Permeability of boundary 1 + ([L] + KD)2

10. THRESHOLD GENERATION with paracrine cycles Rc PARAMETER VALUES

11. PARACRINE AUTOCATALYTIC CYCLES CAN EXPLAIN THRESHOLD GENERATION [eFGF] pM [eFGF] pM %R %R R R Rc

12. [eFGF] pM [eFGF] pM %R %R R R Rc Rc Is receptor dimerization kinetics needed for threshold generation? Receptor dimerization needed for signalling No receptor dimerization

13. Rc Predicted Rc corresponds to experimental observations Rc=100microns~ 100 cells Fraction eFGFRT bound Differentiation signal is related to the percent of bound receptors.

14. EFFECTS OF VARYING PARAMETERS Rc Rc Changing D Changing Ks

15. Rc BIO-ENGINEERING EXPERIMENTS PROPOSED FOR FURTHER STUDY: (Quantitative approach necessary) Measurement of Physiological Concentration Profiles Design a “Microscopic ELISA” Freeze to capture the concentration profile of eFGF. Cryo-microtome  cut thin slices. Thin slices should be fixed fast enough such that Timescale of fixing tissue << Timescale of diffusion of eFGF Detect eFGF with labelled detecting Ab

16. Ks – Rate constant characteristic of the autocrine loop Duplication of eFGF gene and the eFGFR gene with their respective promoters and measure Rc. (stable tranfect clones of their cDNAs driven by their respective promoters) Ks Ks *2 Assuming that the maximal autocrine signalling is limited only by the maximum number of receptors and maximum production of ligand and all intermediates in the signalling cascade are not limiting. Use varying concentrations of inhibitors of eFGFR and measure [eFGF] and [eFGFR] Ks decreased upto zero Study effect of varying system parameters  Identify critical parameters Diffusivity of ECM • Put the cells in matrices with different Diffusivities and measure Rc • Matrigel • HsGAG from cartilage of different species • Synthetic gels embedded with different concentrations of binding Ab

17. Our thoughts on community interactions in development • Autocrine factors that lead to autocatalytic cycles: • EGF, FGF, TGF, VEGF angiogenesis, connective and epithelial tissues, stem cells • IL2, IL13 cells of immune system It is possible that these autocrine cycles lead to important community interactions whenever they occur in cell populations that are in the scale of micrometers to millimeters. e.g. Angiogenesis (VEGF? , EGF?) Maintainence of stem cell number in intestinal crypt ( __F?) Tissue homeostasis At scales of centimeters or higher, community interactions are probably not that important as the effects of convection due to blood flow may predominate.

18. Proposal for further modelling eFGF eFGF gene eFGFR eFGFR gene Xbra gene Driver gene

19. dRT PR KS2[Rb] α[RT] - = + dt D Deff = [RT]KD 1 + ([L] + KD)2 Effect of autocrine loop that increases receptor concentration [eFGF] R Rc Rc

20. Distinction between autocrine loop and autocrine cycles • Recursive • Autocatalytic Autocrine ‘Cycle’ Autocrine Loop In general, difficult to study experimentally Paracrine ‘cycle’

21. Measuring “community factors” can theoretically be used to demonstrate the recursive nature of any autocrine loop Just measure the steady state concentration of community factor attained by cells arranged in a ‘gels’ of different sizes. Can determine Rc, a useful parameter that can describe the overall behaviour of any autocrine / paracrine loop.

22. SUMMARY • Studied community effect in Xenopus myogenesis as a model system. • Proposed and modelled an autocrine-loop mechanism to explain threshold generation in community effect. • Predicted the effect of varying critical parameters on the generated threshold. • Proposed experimental methods to vary these parameters. • Suggested a new method to study and quantitatively describe any autocrine loop.

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