Knockdown of Slit signalling during limb development leads to a reduction in humerus length

Acknowledgements: This project was funded by an EastBio BBSRC DTP PhD Studentship to AR. The authors thank past and present lab staff for helpful discussions.Peer reviewedPostprin

Role of Netrin-1 Signaling in Nerve Regeneration.

Netrin-1 was the first axon guidance molecule to be discovered in vertebrates and has a strong chemotropic function for axonal guidance, cell migration, morphogenesis and angiogenesis. It is a secreted axon guidance cue that can trigger attraction by binding to its canonical receptors Deleted in Colorectal Cancer (DCC) and Neogenin or repulsion through binding the DCC/Uncoordinated (Unc5) A-D receptor complex. The crystal structures of Netrin-1/receptor complexes have recently been revealed. These studies have provided a structure based explanation of Netrin-1 bi-functionality. Netrin-1 and its receptor are continuously expressed in the adult nervous system and are differentially regulated after nerve injury. In the adult spinal cord and optic nerve, Netrin-1 has been considered as an inhibitor that contributes to axon regeneration failure after injury. In the peripheral nervous system, Netrin-1 receptors are expressed in Schwann cells, the cell bodies of sensory neurons and the axons of both motor and sensory neurons. Netrin-1 is expressed in Schwann cells and its expression is up-regulated after peripheral nerve transection injury. Recent studies indicated that Netrin-1 plays a positive role in promoting peripheral nerve regeneration, Schwann cell proliferation and migration. Targeting of the Netrin-1 signaling pathway could develop novel therapeutic strategies to promote peripheral nerve regeneration and functional recovery

Merlin controls the repair capacity of Schwann cells after injury by regulating Hippo/YAP activity

Loss of the Merlin tumor suppressor and activation of the Hippo signaling pathway play major roles in the control of cell proliferation and tumorigenesis. We have identified completely novel roles for Merlin and the Hippo pathway effector Yes-associated protein (YAP) in the control of Schwann cell (SC) plasticity and peripheral nerve repair after injury. Injury to the peripheral nervous system (PNS) causes a dramatic shift in SC molecular phenotype and the generation of repair-competent SCs, which direct functional repair. We find that loss of Merlin in these cells causes a catastrophic failure of axonal regeneration and remyelination in the PNS. This effect is mediated by activation of YAP expression in Merlin-null SCs, and loss of YAP restores axonal regrowth and functional repair. This work identifies new mechanisms that control the regenerative potential of SCs and gives new insight into understanding the correct control of functional nerve repair in the PNS

Visualizing peripheral nerve regeneration by whole mount staining.

Peripheral nerve trauma triggers a well characterised sequence of events both proximal and distal to the site of injury. Axons distal to the injury degenerate, Schwann cells convert to a repair supportive phenotype and macrophages enter the nerve to clear myelin and axonal debris. Following these events, axons must regrow through the distal part of the nerve, re-innervate and finally are re-myelinated by Schwann cells. For nerve crush injuries (axonotmesis), in which the integrity of the nerve is maintained, repair may be relatively effective whereas for nerve transection (neurotmesis) repair will likely be very poor as few axons may be able to cross between the two parts of the severed nerve, across the newly generated nerve bridge, to enter the distal stump and regenerate. Analysing axon growth and the cell-cell interactions that occur following both nerve crush and cut injuries has largely been carried out by staining sections of nerve tissue, but this has the obvious disadvantage that it is not possible to follow the paths of regenerating axons in three dimensions within the nerve trunk or nerve bridge. To try and solve this problem, we describe the development and use of a novel whole mount staining protocol that allows the analysis of axonal regeneration, Schwann cell-axon interaction and re-vascularisation of the repairing nerve following nerve cut and crush injuries

Control of cell shape and plasticity during development and disease by the actin-binding protein Drebrin

Drebrin is an actin-binding protein, originally identified in neuronal cells, involved in the regulation of actin filament organisation, especially during the formation of neurites and cell protrusions of motile cells. Drebrin is found in diverse non-neuronal cells, primarily in association with cell processes and intercellular junctions where it again plays a key role in actin remodelling. The downregulation of Drebrin in Alzheimer’s Disease and Down Syndrome and conversely its upregulation in various carcinomas indicate that Drebrin is an important component of the pathogenesis of multiple diseases

Whole mount staining of transected sciatic nerve at 5 and 7 days after injury.

<p>White arrows mark the proximal (left) and distal (right) nerve stumps and the red arrow within the proximal part of the nerve shows the limit of antibody penetration within this part of the nerve. A and B: whole mount stain of nerve preparations using neurofilament (NF) and S100β (S100) antibodies at 5 days (A) and 7 (B) days after nerve transection. C and D: higher magnification pictures of neurofilament and S100β stain showing interaction of distal Schwann cells (indicated by yellow arrow) with axons.</p

Partnership of Schwann cells and axons during regeneration.

<p>Neurofilament (NF; green), S100β (S100; red) and Hoechst (blue) labelling of axons and Schwann cells at times shown after sciatic nerve transection. A-E: the front edges of the regenerating axons are covered by Schwann cell processes at 4, 5 and 7 days (d). At days 4 and 5, Schwann cell processes (red) form a ‘ball-like’ structure at the tip of axon bundles (A, B and E) whereas at 7d (C and D), fine Schwann cell processes appear to proceed in front of the axons. D: higher magnification of boxed area shown in panel C to show Schwann cell leading processes proceeding in front of axons and guiding axons across the nerve bridge. E and F: higher magnification from the boxed area of panel B. G: higher magnification of boxed area in panel F showing the axonal bundles, white arrows in panel G indicate apparent individual axons. Red arrows in D and H show elongated Schwann cell bodies held by axons crossing the nerve bridge. Upon further axon growth in panel H at 10d, elongated Schwann cell bodies can clearly be seen held by axons in the nerve bridge. In all the panels, the proximal side is up and distal to the bottom of the picture.</p