Edinburgh Explorer Heparan sulphate sulphation by Hs2st restricts astroglial precursor somal translocation in developing mouse forebrain by a non cell autonomous mechanism

Heparan sulphate (HS) is a cell surface and extracellular matrix carbohydrate extensively modified by differential sulphation. HS interacts physically with canonical fibroblast growth factor (FGF) proteins that signal through the extracellular signal regulated kinase activated kinase (MAPK) pathway. At the mouse midline FGF/ERK signalling drives astroglial precursor somal translocation from the ventricular zone of the cortico-septal boundary (CSB) to the induseum griseum (IG) producing a focus of Slit2 -expressing astroglial guidepost cells essential for inter-hemispheric corpus callosum (CC) axon navigation. Here we investigate the cell and molecular function of a specific form of HS sulphation, 2-O HS sulphation catalysed by the enzyme Hs2st, in midline astroglial development and in regulating FGF protein levels and interaction with HS. Hs2st -/- embryos of either sex exhibit a grossly enlarged IG due to precocious astroglial translocation and conditional Hs2st mutagenesis and ex vivo culture experiments show that Hs2st is not required cell autonomously by CC axons or by the IG astroglial cell lineage but rather acts non cell autonomously to suppress the transmission of translocation signals to astroglial precursors. Rescue of the Hs2st -/- astroglial translocation phenotype by pharmacologically inhibiting FGF signalling shows the normal role of Hs2st is to suppress FGF-mediated astroglial translocation. We demonstrate a selective action of Hs2st on FGF protein by showing that Hs2st (but not Hs6st1 ) normally suppresses the levels of Fgf17 protein in the CSB region in vivo and use a biochemical assay to show Hs2st (but not Hs6st1 ) facilitates physical interaction between Fgf17 protein and HS. cell autonomous mechanism. We further show that Hs2st modification 57 selectively facilitates physical interaction between Fgf17 and HS and supresses Fgf17 protein 58 levels in vivo , strongly suggesting that Hs2st acts selectively on Fgf17 signalling. HS 59 interacts with many signalling proteins potentially encoding numerous selective interactions 60 important in development and disease so this class of mechanism may apply more broadly to 61 other biological systems.

protein by showing that Hs2st (but not Hs6st1) normally suppresses the levels of Fgf17 48 protein in the CSB region in vivo and use a biochemical assay to show Hs2st (but not Hs6st1) 49 facilitates physical interaction between Fgf17 protein and HS. Significance statement. 52 We report a novel non cell autonomous mechanism regulating cell signalling in developing 53 brain. Using the developing mouse telencephalic midline as an exemplar we show that the 54 specific sulphation modification of the cell surface and extracellular carbohydrate heparan 55 sulphate (HS) performed by Hs2st suppresses the supply of translocation signals to astroglial 56 precursors by a non cell autonomous mechanism. We further show that Hs2st modification 57 selectively facilitates physical interaction between Fgf17 and HS and supresses Fgf17 protein   The heparan sulphate code hypothesis states that different forms of HS sulphation can 116 encode specific instructions (Kreuger et al., 2006;Turnbull et al., 2001). In this study we     BrdU (BrdU + ) when they were in the VZ before the start of the culture and subsequently 166 exited the VZ and migrated towards the midline over the 2 day culture period when the 167 6 cultures were exposed to experimental substances (SU5402, DMSO, Fgf17 protein, or BSA). 168 4 or 6 sections were quantified per slice moving rostrally from the most caudal section in 169 which the GW could be identified on both sides of the section.  In situ Hybridisation: In situ hybridisation was carried out on 10μm frozen sections as 188 previously described (Wallace and Raff, 1999)      between 2 groups was performed with a t-test. Statistical comparison between > 2 groups was 232 performed with ANOVA followed by post-hoc t-test. p < 0.05 was considered significant.

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Hs2st protein is widely expressed in the developing cerebral cortex and at the 235 telencephalic midline. 236 In order to establish potential sites of action of Hs2st in CC development we first 237 examined the distribution of cells expressing Hs2st protein and contributing to developing 238 CC structures using Hs2st immunohistochemistry at E14.5 (Fig 1 A- shown as higher magnification inset with arrows indicating Hs2st + puncta) with the density 248 falling towards the pial surface although Hs2st + puncta were visible. There were many Hs2st + 249 puncta in the VZ of the cerebral cortex ( Fig 1C) and also in the cortical plate ( Fig 1D) 250 indicating that many cortical progenitors and post-mitotic neurones express Hs2st. At E18.5  The Slit2 expressing IG is expanded in Hs2st -/embryos. 270 We previously reported that increased numbers of astroglia at the pial surface of the  Hs2st -/embryos the Slit2 expression domain is greatly expanded at the pial surface along the 279 rostro-caudal axis (Fig 2 B,D,Fexpanded Slit2 expression domain indicated by brackets). 280 We conclude that an expansion of the Slit2 + IG astroglial population makes a major 281 contribution to the Hs2st -/phenotype.

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To mark the Emx1 lineage, tamoxifen was administered to Emx1 CreER embryos 319 harbouring the floxed-stop GFP reporter at E9.5 so that early Emx1 expressing cerebral 320 cortex progenitors and their descendants were rendered GFP + . Examination of the expression 321 of the GFP reporter shows that, as expected, the developing cerebral cortex and CC axons are 322 of Emx1 lineage (Fig. 3G) and that at the midline GFP expression is predominantly located 323 dorsal to the CSB (dashed lines in Fig 3H). Higher magnification shows that there is a sharp 324 boundary between GFP + and GFPcells at the VZ of the CSB (dashed line in Fig 3I). 325 Combining Sox9 and GFP immunostaining reveals the contribution of the Emx1 lineage to 326 Sox9 + cells. Sox9 + ;GFP + cells (arrowheads in Fig 3I) populate the VZ on the cortical side of 327 the boundary with Sox9 + ;GFPcells on the septal side (arrows in Fig 3I) showing that the    Hs2st is not required cell autonomously by CC axons to navigate the midline. 416 The conditional mutagenesis experiments showed that Hs2st has a non cell Abnormally high FGF/ERK signalling causes the Hs2st -/precocious astroglial 441 translocation phenotype. 442 We previously reported a correlation between hyperactive ERK signalling at the CSB 443 and precocious somal translocation of astroglia to the midline in Hs2st -/embryos but we did  Fig 7J). 471 We conclude that the precocious glial translocation phenotype in Hs2st -/embryos is  Hs2st suppresses Fgf17 protein levels. 479 We next sought to identify an FGF protein that is targeted by Hs2st. Despite its well  (Fig 8A 1,2 -H 1,2 ). At E12.5 telencephalic Fgf17 protein is restricted to the CSB 492 region with no obvious difference between WT and Hs2st -/-(compare Fig 8 A 1 ,B 1 to A 2 ,B 2 ).

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By E14.5 there is an expanded Fgf17 protein domain at the CSB of Hs2st -/embryos 494 (compare Fig 8 D 1 ,E 1 to D 2 ,E 2 , * in E 2 marks the expanded Fgf17 protein domain).

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Quantification of Fgf17 immunofluorescence shows a significant ~2 fold increase in Fgf17 496 protein levels in this region of Hs2st -/-CSB (Fig 8W -compare blue and green bars). At 497 E16.5 Fgf17 protein is much closer to detection threshold than at the earlier stages in both 498 genotypes (Fig 8G 1,2 ,H 1,2 ) although the increased protein spread in the mutant persists (* in 499 Fig 8H 2 ) indicating that the Hs2st -/-CSB is exposed to a prolonged overdose of Fgf17 protein 500 spanning E14.5-E16.5. We next examined Fgf17 mRNA at the CSB so see whether the 501 increase in Fgf17 protein in Hs2st -/-CSB was underpinned by altered Fgf17 gene expression.

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There was no evidence for this at E12.5 or E14.5 where Fgf17 mRNA expression pattern 503 remains similar between Hs2st +/+ and Hs2st -/embryos (compare Fig 8 C 1 to C 2 and F 1 to F 2 ), 504 however, the expression domain of Fgf17 mRNA is increased in E16.5 Hs2st -/-CSB 505 (compare Fig 8I 1  mRNA are GFP - (Fig 8 K 1-3 , arrows indicate GFP + cell location). This complementarity 520 between Fgf17 mRNA expressing and Zic4 lineage cells is preserved at the IG ( Fig 8L 1-3 , 521 arrows indicate GFP + cell location). Fgf17 protein predominates at the IG ( Fig 8M) and 522 higher power magnification shows that while Fgf17 protein is barely detectable at the GW 523 ( Fig 8N 1-3 ) there are a number of much higher Fgf17 expressing cells at the IG and these cells 524 are GFPconfirming that they do not belong to the Zic4 lineage ( Fig 8O 1-3 , arrows indicate 525 GFP + cell location). Interestingly, although cells in the GW and IG express comparable levels 526 of Fgf17 mRNA (compare Fig 8K 1 to L 1 ) the expression of Fgf17 protein is much higher in 527 the IG (compare Fig 8N 1 to O 1 ) suggesting a post-transcriptional repression selectively at the 528 GW. Our identification of Hs2st as a repressor of Fgf17 protein levels at this stage makes 529 Hs2st a strong candidate, indeed, closer examination of Hs2st expression using the Hs2st- 530 LacZ reporter shows that Hs2st is expressed in a GW High -IG Low pattern (Fig 8P, also apparent   531 in the Hs2st immunohistochemistry (Fig 1B)), complementary to the GW Low -IG High Fgf17 532 protein distribution. Together these data bolster the idea that Hs2st acts to suppress Fgf17 Exogenously applied Fgf17 phenocopies the Hs2st -/astroglial translocation phenotype. 551 Our data suggest that the Hs2st -/phenotype stems from abnormally high levels of 552 Fgf17 protein at the CSB causing FGF/ERK hyperactivation and precocious somal 553 translocation to the IG. This requires that Hs2st -/-CSB cells are competent to respond to 554 Fgf17 protein and that application of ectopic Fgf17 triggers precocious glial translocation, 555 neither of which have been previously established. We redeployed the CSB ex vivo culture 556 assay (see Fig 7) with the modification that beads soaked in either recombinant Fgf17 protein 557 (Fgf17 treatment) or in BSA (control) were implanted into coronal slices of CSB region on 558 either side of the midline (Fig 9A). WT or Hs2st -/slices implanted with Fgf17 and BSA 559 beads were cultured for 2 hours before processing for Fgf17 (green signal) and pErk (red 560 signal) double immunofluorescence (Fig 9B). In both WT and Hs2st -/cultures Fgf17 protein 561 was detectable adjacent to the edge of the bead (green signal) and this activated ERK 562 phosphorylation in a similar pattern (red signal) with no obvious differences between WT and 563 Hs2st -/indicating that Hs2st -/-CSB tissue is competent to respond to Fgf17 (Fig. 9B top   564 row). The lack of Fgf17 or pERK signal in the BSA control (Fig 9B, bottom row) confirms 565 Fgf17 antibody specificity and that pERK activation was specifically induced by exogenously 566 applied Fgf17. We performed Sox9/BrdU analysis (exactly as described above for the FGFi 567 experiments - Fig 7) to assess the impact of experimentally introduced Fgf17 on astroglial 568 the Fgf17-bead showed many more Sox9 + (red) cells in the IG region (large arrow on right  sulphation in Hs2st -/tissue (Fig 11 A-J, O). We used the Fgf8:Fgfr3 LACE assay to compare 603 the binding of Fgf8 protein to HS in WT and Hs2st -/-CSB tissue at E14.5 and E16.5 to test the 604 hypothesis that the Fgf8:HS physical interaction is insensitive to loss of 2-O HS sulphation in 605 Hs2st -/tissue (Fig 11 K-N, P). 606 In both E14.5 and E16.5 WT tissue the Fgf17:Fgfr1 and Fgf8:Fgfr3 LACE assays 607 produced a strong LACE signal (Fig 11 A,F    Biochemical (LACE) data shows that physical interaction between Fgf17 and HS is implies that Hs2st might also play a cell-autonomous role in the response to Fgf17 protein. 712 We speculate that even if Hs2st -/astroglial precursors are less sensitive to Fgf17 than their requires precise restoration of normal Fgf17 protein levels (so no rescue could reflect 773 technical failure to restore Fgf17 protein levels to normal) and in any case the CC phenotype 774 of Fgf17 -/embryos has not been thoroughly characterised so Hs2st -/-;Fgf17 -/phenotypes 775 may well be problematic to interpret Rubenstein, 2007, 2008). In addition to not 776 being decisive for or against selectivity we note that demonstrating genetic interaction 777 between Hs2st and Fgf17 would not provide insight into whether the interaction was 778 molecularly direct or not, in contrast to biochemical LACE data we present in the current 779 study.

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The closely related 'Fgf8 subfamily' members Fgf17 and