Heparan sulphate proteoglycans (HSPGs) are a family of molecules that are found on the surface of cells or in the extracellular matrix, where they are involved in regulating key signalling events required for normal mammalian brain development. It is thought that specificity of HSPGs for particular signalling processes is encoded by their heparan sulphate (HS) sugar side chains, which can be modified post-translationally to yield huge variation in HS structure. Different sulphation patterns are generated by the action of the heparan sulphate sulfotransferases (HSTs) and sulfatase enzymes, which add or remove sulphate groups to specific positions on residues of the HS side chains. Depending on the expression of these enzymes and the resulting heparan sulphate ‘code’, it is proposed that cells are then able to regulate signals they receive and send in the ligand rich extracellular environment of the developing forebrain. Hs6st1 and Hs2st catalyse 6-O and 2-O HS sulphation, respectively. Following loss of either of these two HSTs, commissural tracts including the corpus callosum fail to develop normally during late mouse embryogenesis. The telencephalic midline environment is perturbed, with a striking mis-positioning of glial cell populations that normally act to guide axons towards the contralateral hemisphere. Too many radial glial cells at the glial wedge (GW) migrate towards the indusium griseum (IG) in mutant embryos. The running hypothesis to explain this phenotype is a change in critical signalling pathways required to set up the correct midline glia environment, such as Fgf8/ERK signalling which has already been identified as up-regulated at the Hs6st1-/- corticoseptal boundary (CSB). In order to further study what changes are occurring at the developing midline of HST-/- embryos compared to WT, we took a hypothesis free approach using RNA-sequencing analysis. RNA extracted from dissected midline regions of WT, Hs2st-/- and Hs6st1-/- mouse embryos at E16.5 was sent for sequencing, and a list of differentially expressed genes obtained. Overall we find few differentially expressed genes at the Hs6st1-/- midline compared to WT. At the Hs2st- /- midline there are a larger number of differentially expressed genes. Following validation studies, we find a significant and specific increase in Fgf17 protein distribution at the CSB of Hs2st-/- embryos compared to WT at E14.5. The results suggest the hypothesis that Hs2st’s normal role is to regulate Fgf17 protein distribution to limit exposure of GW radial glia cells to this translocation signal. When 2-O HS sulphation is lost then in Hs2st-/- embryos, ectopic Fgf17 signalling induces aberrant glia migration which ultimately prevents callosal axons from crossing the telencephalic midline to form the corpus callosum. To test this hypothesis, we used ex vivo slice culture experiments and showed ectopic Fgf17 protein expression is sufficient to trigger precocious translocation of midline glia in WT CSB, phenocopying the glia behaviour of Hs2st-/- embryos. Also consistent with the hypothesis, the Hs2st-/- glia phenotype can be rescued by addition of an FgfR1 inhibitor which reduces number of translocated glia cells. From these results we find for the first time that 2-O sulphated HS plays a remarkably specific role in regulating Fgf17-mediated translocation of midline glia cells at the developing mammalian telencephalic midline.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:735746 |
| Date | January 2017 |
| Creators | Parkin, Hannah M. |
| Contributors | Pratt, Thomas ; Mason, John |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/25701 |
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