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. 2014 Feb 5;34(6):2389-401.
doi: 10.1523/JNEUROSCI.3157-13.2014.

Heparan Sulfotransferases Hs6st1 and Hs2st Keep Erk in Check for Mouse Corpus Callosum Development

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Free PMC article

Heparan Sulfotransferases Hs6st1 and Hs2st Keep Erk in Check for Mouse Corpus Callosum Development

James M Clegg et al. J Neurosci. .
Free PMC article

Abstract

The corpus callosum (CC) connects the left and right cerebral hemispheres in mammals and its development requires intercellular communication at the telencephalic midline mediated by signaling proteins. Heparan sulfate (HS) is a sulfated polysaccharide that decorates cell surface and extracellular matrix proteins and regulates the biological activity of numerous signaling proteins via sugar-protein interactions. HS is subject to regulated enzymatic sulfation and desulfation and an attractive, although not proven, hypothesis is that the biological activity of HS is regulated by a sugar sulfate code. Mutant mouse embryos lacking the heparan sulfotransferases Hs2st or Hs6st1 have severe CC phenotypes and form Probst bundles of noncrossing axons flanking large tangles of midline glial processes. Here, we identify a precocious accumulation of Sox9-expressing glial cells in the indusium griseum region and a corresponding depletion at the glial wedge associated with the formation of Probst bundles along the rostrocaudal axis in both mutants. Molecularly, we found a surprising hyperactivation of Erk signaling in Hs2st(-/-) (2-fold) and Hs6st1(-/-) (6-fold) embryonic telencephalon that was most striking at the midline, where Erk signaling is lowest in wild-types, and a 2-fold increase in Fgf8 protein levels in Hs6st1(-/-) embryos that could underpin Erk hyperactivation and excessive glial movement to the indusium griseum. The tightly linked Hs6st1(-/-) CC glial and axonal phenotypes can be rescued by genetic or pharmacological suppression of Fgf8/Erk axis components. Overall, our data fit a model in which Hs2st and Hs6st1 normally generate conditions conducive to CC development by generating an HS-containing environment that keeps Erk signaling in check.

Keywords: Fgf8; Sox9; axons; glia; heparan sulfate; mapk.

Figures

Figure 1.
Figure 1.
Similar gross organization of neuronal and glial cell bodies at the telencephalic midline in wild-type (AC), Hs2st−/− (DF), and Hs6st1−/− (GI) embryos at E17.5. Shown is double immunofluorescence for Sox9 (green), which is expressed in RGC and glial cell nuclei, and the postmitotic neuronal nuclear markers NeuN (red; A, B, D, E, G, H) or Tbr1 (red; C, F, I). Nuclei in A, D, and G are counterstained with DAPI (blue). In all three genotypes, most Sox9+ cells are located in the VZ and at the midline, where they form a cluster ventral to NeuN+ or Tbr1+ neurons in the IG. In wild-types, the IG Sox9+ cell population forms above the CC axon bundle, whereas in Hs2st−/− and Hs6st1−/− embryos, it is sandwiched between the PBs. Box in A indicates the region shown at higher magnification in B, C, E, F, H, and I. A, D, and G are to same scale; bar in G is 200 μm. B, C, E, F, H, and I are to same scale, bar in I is 100 μm.
Figure 2.
Figure 2.
Variations in the distribution of glial cells at the telencephalic midline in wild-type, Hs2st−/−, and Hs6st1−/− embryos at E17.5. AL, Sox9 immunofluorescence (green) at the telencephalic midline in wild-type (AD), Hs2st−/− (EH), and Hs6st1−/− (IL) embryos in rostral (A,E,I), middle (B,F,J), and caudal (C,D,G,H,K,L) coronal sections through the developing CC region. Boxed region in A indicates the 250 μm × 250 μm counting area encompassing the IG region used to generate data presented in M. M, Graph showing numbers of Sox9+ nuclei in the IG region counted in serial 10 μm sections spaced at 60 μm intervals along the rostrocaudal axis (mean ± SEM for WT, n = 4 and Hs6st1−/−, n = 3; mean only for Hs2st−/−, n = 2). The pink box indicates the position of the CC (in wild-types) or PBs (in the mutants). Note that whereas midline Sox9+ cell numbers increase moving caudally in all genotypes, the rate of increase is dramatically greater in both mutants in association with PBs. D, H, L, Higher-magnification images of GW and IG region of C, G, and K, respectively. Note that in both mutants, there is a thinning of the Sox9+ area at the GW and more Sox9+ cells at the IG compared with the wild-type. Box in D shows positioning of 100 μm wide radial strip used for quantification of numbers of Sox9+ cells in the GW and IG compartments in NP. (NP) Sox9+ cell counts (mean ± SEM, n = 3 for all genotypes) in the whole strip (GW+IG; N), the GW compartment (O), and the IG compartment (P) taken from serial sections along the rostrocaudal axis binned into rostral, medial, and caudal segments (mean for three sections for each bin). *ANOVA p < 0.05 followed by a post hoc Student's t test for mutant versus wild-type comparison. The trend, most apparent caudally, is for more Sox9+ cells at the IG and fewer Sox9+ cells in the GW in both mutants compared with the wild-type. Scale bars: AC, EG, and IK, 200 μm; D, H, and L, 100 μm.
Figure 3.
Figure 3.
Increased migration of glial cells from the GW to the IG in Hs2st−/− and Hs6st1−/− embryos. (AC, EG) BrdU (red) and Sox9 (green) immunofluorescence at the telencephalic midline of WT (A, E), Hs2st−/− (B, F), and Hs6st1−/− (C, G) embryos at E16.5 after a single BrdU administration at E14.5. EG, Higher-magnification images of the IG region in AC, respectively. D, Quantification of the number of Sox9+BrdU+ double-labeled cells (yellow) at the IG within a 250 μm × 250 μm counting area indicated in A. Histogram shows mean ± SEM: WT, n = 4; Hs2st−/−, n = 3; Hs6st1−/−, n = 4. *ANOVA p < 0.05 followed by a post hoc Student's t test for mutant versus wild-type comparison. The number of double-labeled cells at the IG is significantly increased in both Hs2st−/− and Hs6st1−/− embryos compared with WT. HJ, PH3 immunohistochemistry shows similar location of mitotic cells at the VZ of the GW in wild-type (H), Hs2st−/− (I), and Hs6st1−/− (J) embryos and cell counts (K) confirm a similar PH3+ cell density in the GW region of the VZ between genotypes at E16.5. Histogram shows mean ± SEM, n = 3 for all genotypes. Scale bars: AC and HJ, 100 μm; EG, 50 μm.
Figure 4.
Figure 4.
Fgf8 protein and mRNA expression in the telencephalon at E16.5 in wild-type, Hs2st−/−, and Hs6st1−/− embryos. A, Western blot of telencephalic protein and quantification for Fgf8 protein with β-actin protein used for normalization. Histograms shows mean ± SEM for WT, n = 7; Hs2st−/−, n = 4, Hs6st1−/−, n = 3. *ANOVA p < 0.05 followed by a post hoc Student's t test for mutant versus wild-type comparison. BG, Immunofluorescence for Fgf8 (red) on coronal sections. Boxed areas in BD shown at higher magnification in EG. Fgf8 protein levels are much higher at the telencephalic midline of Hs6st1−/− embryos compared with the other genotypes. H, I, COS7 cells transfected with a mouse Fgf8 cDNA expression construct show specific immunofluorescent staining (H), whereas untransfected cells do not (I). J, Fgf8 immunofluorescence is below detection limit in Fgf8neo/neo-matched telencephalic tissue homozygous for a severely hypomorphic Fgf8 allele. K, qRT-PCR analysis of RNA extracted from the medial telencephalon shows no significant difference in Fgf8 mRNA expression between WT and Hs6st1−/− embryos. Histograms shows mean ± SEM for WT, n = 5; Hs6st1−/−, n = 4. L, M, In situ hybridization shows that in both WT (L) and Hs6st1−/− (M) embryos Fgf8 mRNA is similarly localized to the IG and GW and expressed below detection threshold elsewhere in the telencephalon. Scale bars: BG and JM, 200 μm; I and J, 100 μm.
Figure 5.
Figure 5.
Erk signaling in the embryonic telencephalon in wild-type, Hs2st−/−, and Hs6st1−/− embryos at E16.5 (AH) and E17.5 (IN). A, Western blot for pErk1/2 and β-actin (loading control) in protein extracted from whole telencephalon, quantification shows pErk1/2 levels relative to β-actin. pErk1/2 expression is significantly increased in both Hs2st−/− and Hs6st1−/− embryos compared with wild-type. B, Western blot for total Erk1/2, and β-actin (loading control) shows no increase in Erk1/2 expression in either Hs2st−/− or Hs6st1−/− embryos. Histograms in A and B show mean ± SEM for WT, n = 7; Hs2st−/−, n = 4, Hs6st1−/−, n = 3. *ANOVA p < 0.05 followed by a post hoc Student's t test for mutant versus wild-type comparison. CN pErk1/2 immunohistochemistry on (CH) E16.5 and (IN) E17.5 coronal section from wild-type (C, F, I, L), Hs2st−/− (D, G, J, M), and Hs6st1−/− (E, H, K, N) embryos. Arrowheads in CE, demarcate the cerebral cortex from the GW at the CSB (filled arrowhead) to the PSPB (unfilled arrowhead). FH and LN are higher magnifications of the GW area in CE and IK. Both mutants exhibit a global increase in pErk1/2 compared with wild-type, which is particularly striking at the telencephalic midline, where levels are relatively low in wild-type embryos. Within the GW, a larger number of pErk1/2-positive cells (examples indicated by arrows) can be seen in the Hs2st−/− (G, M), which is even more pronounced in the Hs6st1−/− (H, N) compared with WT (F, L). Scale bars: CE and IK, 200 μm; FH and LN, 20 μm.
Figure 6.
Figure 6.
Lef1 and pSmad1/5 protein expression in the embryonic telencephalon of wild-type, Hs2st−/−, and Hs6st1−/− embryos at E16.5. A, Western blots for Lef1, pSMAD1/5, and β-actin (loading control) in protein extracted from whole telencephalon. Quantification shows relative protein expression relative to β-actin level. Note that Lef1 and pSMAD1/5 were quantified in separate blots, which were each simultaneously probed with β-actin. Lef1 expression appears unchanged in both Hs2st−/− and Hs6st1−/− embryos compared with wild-type. pSMAD1/5 expression is slightly increased in Hs2st−/− embryos compared with wild-type and is significantly increased in Hs6st1−/− embryos compared with wild-type. Histograms shows mean ± SEM for WT, n = 4; Hs2st−/−, n = 4, Hs6st1−/−, n = 4. *ANOVA p < 0.05 followed by a post hoc Student's t test for mutant versus wild-type comparison. BG, Immunohistochemistry for Lef1 (B, D, F) and pSMAD1/5 (C, E, G) at E16.5 on coronal section of wild-type (B, C), Hs2st−/− (D, E), and Hs6st1−/− (F, G) telencephalon. The expression pattern of both Lef1 and pSMAD1/5 at the telencephalic midline appears similar in WT, Hs2st−/−, and Hs6st1−/− embryos. Scale bar, 200 μm.
Figure 7.
Figure 7.
Rescue of the Hs6st1−/− CC phenotype by repressing the Fgf8/Erk axis. AD, Reducing Fgf8 gene dosage ameliorates the Hs6st1−/− CC phenotype. AC, Hs6st1−/−;Fgf8+/+ (A) and Hs6st1−/−;Fgf8+/− (B,C) embryos in which B shows a completely unrescued and C a completely rescued Hs6st1−/− phenotype. D, WT (Hs6st1+/+;Fgf8+/+) control embryo. χ2 = 4.13, df = 1; p < 0.05. EN, MEKi treatment ameliorates the Hs6st1−/− CC phenotype. Hs6st1−/− embryos from Hs6st1+/− × Hs6st1+/− crosses from uninjected (E), vehicle-injected (F), and MEKi-injected dams (G, H), in which H shows a completely unrescued and G a completely rescued Hs6st1−/− phenotype. χ2 = 6.55, df = 1; p < 0.05. AH show immunofluorescence for the axonal marker L1 (red) and the glial marker GFAP (green) in coronal sections at E18.5. Numbers at the bottom left indicate the proportions of embryos with phenotype shown in that panel. IN, Sox9 immunofluorescence at E18.5 on wild-type uninjected embryos (I), Hs6st1−/− vehicle-injected embryos with severe Hs6st1−/− CC phenotype (J), and Hs6st1−/− MEKi-injected embryos showing a rescue of the CC phenotype (K). Box in I shows positioning of 100-μm-wide radial strip used for quantification of numbers of Sox9+ cells in the GW and IG compartments in LN. LN, Sox9+ cell counts in the whole strip (GW+IG; L), the GW compartment (M), and the IG compartment (N) taken from serial sections along the rostrocaudal axis binned into rostral, medial, and caudal segments (mean for three sections for each bin). Plots shows mean ± SEM, n = 3 for all conditions. *ANOVA p < 0.05 followed by a post hoc Student's t test for MEKi (rescue) versus vehicle comparison. The total number of Sox9+-stained cells along the whole strip is similar in the WT, vehicle, and MEKi groups (L). Sox9+ cell number at the GW region is reduced in the vehicle-injected Hs6st1−/− embryos, but is returned to WT level in rescued MEKi-treated Hs6st1−/− embryos (M). Sox9+ cell number at the IG is increased in vehicle-injected Hs6st1−/− embryos, but is returned to WT level in rescued MEKi treated Hs6st1−/− embryos (N). Scale bars, 200 μm.
Figure 8.
Figure 8.
Model summarizing cellular and molecular phenotypes of Hs2st−/− and Hs6st1−/− embryos at the telencephalic midline. A, In WT embryos, Hs6st1 and (to a lesser extent) Hs2st activities both repress the Erk signaling pathway to maintain a level of pErk that drives appropriate numbers of Sox9+ glia (green) to translocate (pink arrows) from the GW to the IG and generates a GW ↔ IG glial balance that guides CCAs (red arrow) across the telencephalic midline in 100% of embryos. Loss of Hs2st (B) or Hs6st1 (C) activity results in a derepression of the Erk pathway, excess levels of pErk in the GW, and excess GW → IG translocation of Sox9+ glia-blocking (red X) CCAs from crossing the telencephalic midline. The Hs2st−/− and Hs6st1−/− phenotypes differ quantitatively, with Hs2st−/− embryos exhibiting 2-fold Erk hyperactivation and ∼50% of embryos developing a normal CC and Hs6st1−/− embryos exhibiting 6-fold Erk hyperactivation and 0% developing a normal CC. D, Repressing the hyperactive Erk pathway in Hs6st1−/− embryos restores the GW ↔ IG glial balance and ameliorates the severity and penetrance of the CC phenotype, with ∼33% of embryos developing a normal CC using the treatments used in this study. Thickness of “T” symbol indicates repressor strength and size of pErk text indicates pErk levels in each genotype. Numbers at bottom indicate proportion (%) of embryos of each condition that produce a normal CC.

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