Characterisation of the pro-regenerative extracellular matrix in the zebrafish spinal cord

Turnover and modification of the extracellular matrix (ECM) plays a fundamental role in the repair and regeneration of wounds to tissues throughout the body, including spinal cord injuries (SCIs). In this thesis I present a functional study of the effects of core ECM components on neuronal and glial cell behaviour in two models of SCI: the highly-regenerative larval zebrafish; and an in vitro model of the non-regenerative rat (MC-Inj). Tests using heparin-derived mimetics (mHeps) of heparan sulphate proteoglycans (HSPGs) indicate that a densely-sulphated glycan structure shown to be anti-regenerative in MC-Inj (mHep1) stimulates axonal regeneration in the zebrafish. A screen of specifically modified variations on this structure revealed that the responses of injured axons and glia to the extracellular HS they encounter are directly influenced by the precise arrangement of sulphate groups on the HS chains. Exposure to an mHep variant with desulphated GlcNAc residues (mHep2) caused anti-regenerative effects in the zebrafish, but no effects in MC-Inj. My analysis of the effects of candidate mHeps from this screen on the behaviour of GFAP-positive spinal cord glia highlighted differences in the relationship between the neuronal and glial regeneration programmes in zebrafish versus rats. Additionally, I began an investigation into the role of the collagenous ECM in zebrafish SCI regeneration using a mutant line lacking the collagen-binding matricellular glycoprotein Sparc. Surprisingly, offspring from the stable sparc-null zebrafish line do not show the spinal cord regeneration impairment noted in the acutely CRISPR-injected generation. Further, I present the optimisation of a proteomic protocol for the analysis of ECM proteins enriched from adult zebrafish spinal cord tissue. This can be adapted to the study of the broader effects of matrisome gene knockouts or injuries on the spinal cord ECM in this highly-regenerative model species. The data presented here help to characterise the gap in SCI regenerative capacity between zebrafish and mammals. The results implicate not only fundamental species differences in the interpretation of the injury site ECM by local cells, but also the relationship between neuronal and glial cell types during regeneration.

Grantor dc:publisher

The University of Edinburgh

Year dc:date.issued

2024

Author dc:creator

• Lake, Elizabeth Jesse

Advisors dc:contributor.advisor

• Sieger, Dirk
• Becker, Catherina
• Becker, Thomas
• Barnett, Susan

• zebrafish
• spinal cord injury
• ECM proteins
• proteoglycans
• heparan sulphate proteoglycans
• HSPGs
• mHeps
• regenerative capacity

Language dc:language.iso

en

Identifier URI

http://dx.doi.org/10.7488/era/4425

OAI identifier oai:identifier

oai:era.ed.ac.uk:1842/41702