430 Seminar - A Love-Hate Relationship

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A ‘Love-Hate’ Relationship: How Eph/Ephrin signaling can be both attractive and repulsive : 

A ‘Love-Hate’ Relationship: How Eph/Ephrin signaling can be both attractive and repulsive By Darren Devison & Dan Hynes Regulation of Repulsion Versus Adhesion By Different Splice Forms of an Eph Receptor (Holmberg et al)

Seminar Overview : 

Seminar Overview Background Experiments & Results Discussion

Background : 

Background

Background : 

Background Eph Receptor Tyrosine Kinases Membrane bound receptors Interact with membrane bound ligands, ephrins Largest family of RTKs

Background : 

Background 14 receptors discovered thus far in vertebrates GPI-linked Transmembrane 1 receptor discovered in invertebrates GPI-linked

Background : 

Background Previous work has shown that eph signaling is needed for: Neuronal development Angiogenesis Spatial boundaries between tissues Embryonic development and morphogenesis

Background : 

Background It was not always this clear a picture

Background : 

Background Eph/Ephrin signaling thought to be repulsive (with some exceptions) Neural fold defects in Ephrin-A5 mutants indicated adhesion role

Background : 

Background Paper sought to show: EphA7 has alternative splice variants that are truncated prior to the tyrosine kinase domain Dimerization of EphA7-T1 (truncated isoform) with EphA7-FL (full length isoform) resulted in adhesion That adhesion was not the result of suppressing repulsion, but was a signalling phenomenon of its own Add to the growing knowledge regarding abnormal brain development, as it may be applied to humans

Experiments& Results : 

Experiments& Results

Experiments/Results : 

Experiments/Results Null mutants can complete neural fold shown by d) - 17% Ephrin-A5 null mutants show failure of neural folds to close shown by a),b)

Experiments/Results : 

Experiments/Results Cell proliferation continues when fusion does not occur Neural folds turn outward 71% affected were female

Experiments/Results : 

Experiments/Results In situ hybridization showed expression of ephrin-A5 (g)

Experiments/Results : 

Experiments/Results - Embryos with Ephrin-A5-Fc failed to show expression at 10 somite stage (h) - Strong expression show during fusion (m)

Experiments/Results : 

Experiments/Results Expression of EphA7-Fc was shown at the neural folds

Experiments/Results : 

Experiments/Results RT-PCR was performed on edge of cranial folds Primers for all known EphA receptors Showed only EphA7 in three forms Full length EphA7 and two truncated forms Truncated forms lacked kinase domains - In situ hybridization and immuno-histochemistry confirmed expression on neural folds

Experiments/Results : 

Experiments/Results Effect of ephrin-A5 was observed by changing gene expression Then observing adhesions ability in wells of EphA7-Fc (a) (b) was used to show proper protein expression in stable lines

Experiments/Results : 

Experiments/Results - (c) Shows migration of cells expressing EphA7 towards ephrin-A5-Fc - Cells expressing EphA7-FL were significantly repelled - Cells expressing both EphA7-FL and EphA7-T1 were not significantly repelled

Experiments/Results : 

Experiments/Results B – similar levels of expression for FL and T1 in different somite stages C – model for repulsive and adhesive properties of receptor/ligand complex in both FL and T1 scenarios

Experiments/Results : 

Experiments/Results Co-IP elutant (EphA7) blotted for tyrosine activity (i.e. pTyr) Co-IP elutant (EphA7) blotted for presence of elutant (EphA7) EphA7-FL mRNA (via RT-PCR), shows expression levels of this receptor EphA7-T1 mRNA expression levels via RT-PCR (higher with higher Dox) Loading Control 1 2 3 4 5

Experiments/Results : 

Experiments/Results Receptor-expressing cells after aggregation with ephrin-A5-expessing cells - FACs (fluorescence activated cell sorting) FACs analysis FACs analysis Photomicrograph Photomicrograph Uninduced Doxycyclin-induced

Experiments/Results : 

Experiments/Results A – EphA7-FL (red) avoid ephrin-A5-expressing cells (green) B – With EphA7-T1 induced, EphA7-FL grows closely with ephrin A5 cells A B

Experiments/Results : 

Experiments/Results C,D – Uninduced (C) or doxycyclin-induced (D) receptor expressing cells - Plated on preformed confluent mosaics of wild-type 293 cells (yellow) and ephrin-A5-expressing cells (green) C D

Experiments/Results : 

Experiments/Results EphA7 FL cells had preference for growth on wild-type cells 2. Co-expressing cells (EphA7 FL/ EphA7 T1) preferred growth on ephrinA5-expressing cells 3. EphA7 T1 cells grew predominantly on ephrin-A5-expressing cells

Discussion : 

Discussion

Discussion : 

Discussion Similarities to anencephaly in humans EphA7 main functional receptor for ephrin-A5 Area of expression reason for localized defect Three different splice forms of EphA7

Discussion : 

Discussion - Truncated EphA7 has increased affect on adhesion - repulsion mechanism involves phosphorylation of kinase domain Co-expression of EphA7-T1 and EphA7-FL changes amount of phosphorylation - EphA7-T1 negative suppression of repulsion

References : 

References Brooksbank, C. 2000. Cell Adhesion: How to lead a double life. Macmillan Magazines. 1. Davy, A., and Robbins, S. 2000. Ephrin-A5 modulates cell adhesion and morphology in an integrin-dependent manner. The Embo Journal. 19(20): 5396-5405. Flanagan, J., and Vanderhaeghen, P. 1998. The ephrins and eph receptors in neural development. Annu. Rev. Neurosci. 21:309-345. Frisen, J., Holmberg, J., and Barbacid, M. 1999. Ephrins and their Eph receptors: multitalented directors of embryonic development. The EMBO Journal. 18(19): 5159-5165. Holder, N., and Klein, R. 1999. Eph receptors and ephrins: effectors of morphogenesis. Development. 126: 2033-2044. Holmberg, J., Clarke, D., and Frisen, J. 2000. Regulation of repulsion versus adhesion by different splice forms of an Eph receptor. Nature. 408: 203-206 Huai, J., and Drescher, U. 2001. An eprhin-A-dependent Signaling Pathway Controls Integrin Function and Is Linked to the Tyrosine Phosphorylation of a 120-kDa Protein. Journal of Biological Chemistry. 276(9):6689-6694. O’Leary, D., and Wilkinson, D. 1999. Eph receptors and ephrins in neural development. Current Opinion in Neurobiology. 9:65-73.

Questions? : 

Questions?