Heparan Sulfate Sulfation by Hs2st Restricts Astroglial Precursor Somal Translocation in Developing Mouse Forebrain by a Non-Cell-Autonomous Mechanism

Hs2st protein is widely expressed in the developing cerebral cortex and at the telencephalic midline

To establish potential sites of action of Hs2st in CC development, we first examined the distribution of cells expressing Hs2st protein and contributing to developing CC structures using Hs2st immunohistochemistry at E14.5 (Fig. 1A–D) and E18.5 (Fig. 1E–M) spanning the period of CC axon tract development. Macroscopically, Hs2st protein distribution closely resembles the Hs2st-LacZ reporter staining previously reported, with widespread Hs2st expression in the developing cerebral cortex and at the CSB at both E14.5 and E18.5 (Fig. 1A,E, boxed areas indicate regions shown at higher magnifications in B–D and F–M) (Conway et al., 2011). Subcellularly, the Hs2st signal is punctate consistent with the expected localization of Hs2st in the Golgi apparatus (arrows point to Hs2st⁺ puncta in higher-magnification insets in Fig. 1B,F,P). At E14.5, there was a high density of Hs2st⁺ puncta at the CSB in the GW region where IG astroglial RGC prescursors reside (Fig. 1B, with boxed area shown as higher-magnification inset with arrows indicating Hs2st⁺ puncta), with the density falling toward the pial surface although Hs2st⁺ puncta were visible. There were many Hs2st⁺ puncta in the VZ of the cerebral cortex (Fig. 1C) and also in the cortical plate (Fig. 1D), indicating that many cortical progenitors and postmitotic neurons express Hs2st. At E18.5, Hs2st is expressed by many cells in the IG (Fig. 1F) and at the apical surface of the VZ at the GW (Fig. 1G), septum (Fig. 1H), and ventral telencephalon (Fig. 1I), with the number of Hs2st expressing VZ cells diminishing as distance from the ventricle increases. In the cerebral cortex, Hs2st is expressed by many cells close to the apical surface of the VZ (Fig. 1J). Large numbers of postmitotic cortical neurons outside of the VZ express Hs2st and, moving toward the pial surface, the density of Hs2st⁺ puncta varies with laminar position [cf. Fig. 1K–M, showing relatively high Hs2st⁺ puncta density in cortical layers adjacent to the pial membrane (M) and in the intermediate zone (K) and lower density in the intervening region (L)]. We validated the Hs2st antibody by demonstrating absence of the punctate Hs2st⁺ immunostaining in Hs2st^−/− embryonic material (cf. Fig. 1N,P and O,Q; note that the more diffuse staining persists in Hs2st^−/− tissue and we discounted this as nonspecific background) and Western blot showing that the predicted 42 kDa Hs2st protein band was present in Hs2st^+/+ and absent from Hs2st^−/− telencephalic protein extracts (Fig. 1R). To conclude, Hs2st protein is present in developing cerebral cortex, the source of CC axons, as well as in progenitor and postmitotic cells of the CSB region constituting the environment through which midline crossing CC axons navigate. Hs2st expression analysis suggests multiple potential sites of action for 2-O HS sulfation in CC development.

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Figure 1.

Hs2st protein is expressed in the cerebral cortex and the septum during CC formation. A, Immunohistochemistry for Hs2st at E14.5. B–D, Higher magnification showing punctate subcellular Hs2st expression (inset, B) Hs2st protein is expressed at the CSB (B), the VZ of the cortex (C), and the cortical plate (D). E, Immunohistochemistry for Hs2st at E18.5 (F–M) Hs2st protein is expressed in the IG (F), the GW (G), the septum (H), and the ventral telencephalon (I). Within the cortex, Hs2st is expressed at the VZ (J) and the intermediate zone (K); Hs2st is not strongly expressed by the middle layers of the cortex (L), but is expressed by the deeper layers (M). N–R, Hs2st antibody specificity. The Hs2st antibody produces signal in the GW (N, P), which is lost in Hs2st^−/− embryos (O, Q). Western blot performed on protein extracted from whole telencephalon using Hs2st antibody revealed the predicted ∼42 kDa band in WT extracts, which is lost in Hs2st^−/− extracts (R). B–D are higher-magnification images of boxed regions indicated in A. F–M are higher-magnification images of boxed regions indicated in E. P and Q are higher-magnification images of boxed regions in N and O, respectively. Insets in B, F, P and Q show higher magnification with arrows indicating Hs2st puncta. Scale bars: A, 500 μm; B–I, L, M, 50 μm; J, K, 100 μm.

Slit2-expressing IG is expanded in Hs2st^−/− embryos

We previously reported that increased numbers of astroglia at the pial surface of the Hs2st^−/− CSB stemmed from precocious glial translocation and found no evidence that changes in cell proliferation or death contributed to this phenotype (Conway et al., 2011; Clegg et al., 2014). To determine whether there is an expansion of the IG in Hs2st^−/− embryos, we compared the expression of Slit2 mRNA, a marker of GW and IG glia but not MZ glia, between Hs2st^+/+ and Hs2st^−/− embryos at E16.5 (Shu and Richards, 2001; Shu et al., 2003). In Hs2st^+/+ embryos Slit2⁺ cells form a compact focus at the IG that increases in size moving caudally (Fig. 2A,C,E, Slit2 expression domain at IG indicated by brackets). In Hs2st^−/− embryos, the Slit2 expression domain is greatly expanded at the pial surface along the rostrocaudal axis (Fig. 2B,D,F, expanded Slit2 expression domain indicated by brackets). We conclude that an expansion of the Slit2⁺ IG astroglial population makes a major contribution to the Hs2st^−/− phenotype.

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Figure 2.

Slit2 expression at the CSB of WT and Hs2st^−/− embryos at E16.5. A, C, E, In situ hybridization for Slit2 in WT embryos at 3 rostrocaudal positions labeling the GW and IG. B, D, F, In situ hybridization for Slit2 in Hs2st^−/− embryos at equivalent positions to A, C, and E, respectively, showing an expanded IG. Scale bars, 100 μm in all panels.

Cell autonomy of HS and 2-O HS sulfation in astroglial precursor somal translocation and CC development

We next exploited conditional mutagenesis of Hs2st or Ext1 to experimentally uncouple specific functions of 2-O sulfation from more general functions of HS in astroglial precursor translocation and CC development. Widespread expression of HS and 2-O HS sulfation leaves open the possibility that each regulates GW→IG astroglial precursor somal translocation cell autonomously by modulating the response to signals, non-cell autonomously by regulating the supply of signals, or both. To resolve this, we identified two Cre alleles, Zic4^Cre and Emx1^CreER, that drive LoxP-mediated mutagenesis in the astroglial lineage or in their cellular environment, respectively, and used them to conditionally ablate either HS (Ext1^LoxP mutagenesis) or 2-O HS (Hs2st^LoxP mutagenesis) sulfation to test for cell-autonomous or non-cell-autonomous functions. We refer to these as “Zic4 lineage” and “Emx1 lineage,” respectively, and next present their characterization using a floxed-stop GFP reporter that turns on GFP expression in Cre-expressing cells and their descendants before describing experiments where they are used to conditionally generate loss-of-function mutations in Ext1^Fl or Hs2st^Fl alleles (Inatani et al., 2003; Kessaris et al., 2006; Rubin et al., 2010; Sousa et al., 2009; Stanford et al., 2010).

Characterization of Zic4 and Emx1 lineages

The septum is of Zic4 lineage, as shown by strong expression of the GFP reporter (Fig. 3A). The GFP signal in the intermediate zone of the cerebral cortex (asterisks in Fig. 3A) is due to GFP⁺ thalamocortical axons that project from Zic4-lineage cells in the thalamus and cells of subcortical origin as described previously (Rubin et al., 2010). At the midline, GFP⁺ cells of the Zic4 lineage are predominantly located ventral to the CSB (dashed lines in Fig. 3B), but there is also GFP expression in the IG (boxed area “D” in Fig. 3B). Sox9 is a transcription factor that marks the nuclei of RGCs in the VZ and differentiated astroglia in the IG and MZG and we previously showed that the positioning of Sox9⁺ cells is of critical importance for the development of the CC (Clegg et al., 2014). Combining GFP with Sox9 immunostaining reveals the contribution of the Zic4 lineage to the CSB astroglial populations. There is a sharp boundary (dashed line in Fig. 3C) in the VZ of the CSB between Sox9⁺;GFP⁺ cells (arrowheads in Fig. 3C) on the septal side and Sox9⁺;GFP⁻ cells (arrows in Fig. 3C) on the cortical side. Virtually all the Sox9⁺ cells in the IG (Fig. 3D) and MZG (Fig. 3E) are also GFP⁺ (arrowheads in Fig. 3D,E), indicating that these cells are of the Zic4 lineage. These data show that the Zic4 lineage contributes Sox9⁺ cells to the septal VZ and, strikingly, is the sole source of Sox9⁺ astroglia in the IG (Fig. 3F).

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Figure 3.

Emx1 and Zic4 lineage contribution at the CSB. A, Zic4^cre allele combined with a lox-stop GFP reporter has been used to label cell populations at E18.5. Zic4^cre labels cells of the septum (asterisks in 3A mark GFP in cortex). B–E, Zic4^cre labels cells ventral to the CSB (dashed line, B,C) including Sox9 expressing cells (arrowheads, C), but is not expressed by Sox9 expressing cells dorsal to the CSB (arrows, C). Zic4^cre is expressed by IG glial cells (arrowheads, D) but not by surrounding cells. Zic4^cre is expressed by MZ glial cells (arrowheads, E). F, Schematic of the Zic4^cre expressing cell lineage. G, Emx1^creER allele combined with a lox-stop GFP reporter has been used to label cell populations at E18.5. Emx1^creER labels cells of the cortex. H–K, Emx1^creER labels cells dorsal to the CSB (dashed line, H, I) including Sox9 expressing cells (arrowheads, I), but is not expressed by Sox9-expressing cells ventral to the CSB (arrows, I). Emx1^creER is not expressed by IG glial cells (arrows, J). Emx1^creER is not expressed by MZ glial cells (arrows, K). L, Schematic of the Emx1-expressing cell lineage. No phenotype was detected in Hs2st^+/+ Zic4^cre or Hs2st^+/+ Emx1^creER embryos (n = 5 for each genotype). C–E are higher-magnification images of the indicated regions in B. I–K are higher-magnification images of the indicated regions in H. Scale bars: A, G, 500 μm; B, H, 200 μm; C–E and I–K, 50 μm.

To mark the Emx1 lineage, tamoxifen was administered to Emx1^CreER embryos harboring the floxed-stop GFP reporter at E9.5 so that early Emx1 expressing cerebral cortex progenitors and their descendants were rendered GFP⁺. Examination of the expression of the GFP reporter shows that, as expected, the developing cerebral cortex and CC axons are of Emx1 lineage (Fig. 3G) and that, at the midline, GFP expression is predominantly located dorsal to the CSB (dashed lines in Fig. 3H). Higher magnification shows that there is a sharp boundary between GFP⁺ and GFP⁻ cells at the VZ of the CSB (dashed line in Fig. 3I). Combining Sox9 and GFP immunostaining reveals the contribution of the Emx1 lineage to Sox9⁺ cells. Sox9⁺;GFP⁺ cells (arrowheads in Fig. 3I) populate the VZ on the cortical side of the boundary with Sox9⁺;GFP⁻ cells on the septal side (arrows in Fig. 3I), showing that the Emx1 lineage contributes Sox9⁺ cells exclusively to the cortical side of the VZ. All Sox9⁺ cells in the IG (Fig. 3J) and MZ (Fig. 3K) are GFP⁻ (arrows in Fig. 3J,K), indicating that the Emx1 lineage does not contribute Sox9⁺ cells to the IG. These data show that the Emx1 lineage contributes Sox9⁺ cells to the cortical VZ, but no cells of this lineage contribute Sox9⁺ astroglia to the IG (schema in Fig. 3L).

Ext1 is required by both Emx1- and Zic4-lineage cells for CC development

To determine the cellular requirement for HS, we deleted Ext1, which is essential for HS synthesis, in the Zic4 or Emx1 lineages. In control embryos, L1 immunostaining labels axons in the U-shaped CC, whereas GFAP staining labels midline astroglial structures (Fig. 4A, with higher magnification of IG and GW in D,G). Removing HS from either the Zic4 lineage (Fig. 4B, with higher magnification of IG and GW in Fig. 4E,H) or the Emx1 lineage (Fig. 4C, with higher magnification of IG and GW in Fig. 4F,I) generates a severe CC agenesis phenotype (Zic4^Cre;Ext1^Fl/Fl n = 4/4; Emx1^CreER;Ext1^Fl/Fl n = 3/3). In Emx1 conditional mutants (Emx1^CreER;Ext1^Fl/Fl), CC axons fail to cross the midline and form Probst bundles short of the midline (Fig. 4B). GFAP⁺ astroglial cells are present in the IG (Fig. 4E) and at the GW (Fig. 4H) in a pattern grossly similar to that of controls (cf. Fig. 4D,G and E,H). In Zic4 conditional mutants (Zic4^Cre;Ext1^Fl/Fl), CC axons approach the midline but fail to cross (Fig. 4C, with higher magnification of IG and GW in F,I). Astroglial populations in Zic4 conditional mutants are obviously disrupted, with less intense GFAP staining at the midline (cf. IG region in Fig. 4D,F) and more GFAP at the GW than in controls (arrows in Fig. 4I; cf. Fig. 4G,I), suggesting that, in these embryos, astroglial precursors translocate less efficiently to the IG and instead remain in the GW. The cerebral cortex of Zic4^Cre;Ext1^Fl/Fl brains was thinned and the ventricles were enlarged (cf. Fig. 4A,C) and this hydrocephalus-like phenotype is intriguing because the cerebral cortex is not of the Zic4 lineage, indicating a non-cell-autonomous mechanism by which HS regulates cerebral cortex development. The FGFR1/FGF2 LACE assay detects endogenous HS on tissue sections by forming ternary complexes with exogenously added FGF2 and FGFR1 (red LACE signal in Fig. 4J–O) (Allen et al., 2001; Chan et al., 2015). HS is ubiquitously expressed in both cortical and septal compartments of control telencephalon (Fig. 4J, higher magnification of CSB in M) and, as intended, HS synthesis is blocked in the cortex and cortical axons of Emx1^CreER;Ext1^Fl/Fl embryos (Fig. 4K, CSB shown at higher magnification in N, with arrows indicating HS-deficient cortical region) and in the septum of Zic4^Cre;Ext1^Fl/Fl embryos (Fig. 4L, higher magnification of CSB in O, with arrows indicating HS-deficient septum). Predigesting tissue sections with heparitinase eliminated the LACE signal (data not shown), confirming the specificity of this assay for detecting HS. The salient conclusions from the Ext1 conditional mutagenesis for the current study are that HS is indispensable from both the Zic4 and the Emx1 lineages for CC development and that removing HS from the Zic4 lineage inhibits Zic4 lineage astroglia from reaching the IG region.

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Figure 4.

HS expression is required within both Emx1 and Zic4-lineage cells for CC formation. A–I, Immunofluorescence for L1 (red) at E18.5 labels the CC, whereas GFAP (green) labels glia. In control embryos, the U-shaped CC has formed and is flanked by glia at the IG and GW (A, D, G). In Ext1^fl/fl Emx1^creER embryos, CC axons do not cross the midline, whereas glia at the IG and GW appear largely unaffected (B, E, H). In Ext1^fl/fl Zic4^cre embryos, CC axons do not cross the midline, whereas glia appear depleted at the IG and form abnormal bundles at the GW (C, F, I). J–O, FGFR1/FGF2 LACE assay is used to detect the presence of HS. In control embryos, the LACE signal can be seen throughout the telencephalon and is of similar intensity within both the cortex and the septum (J, M). In Ext1^fl/fl Emx1^creER embryos, the LACE signal is significantly reduced within the cortex (K, N). In Ext1^fl/fl Zic4^cre embryos, the LACE signal is significantly reduced within the septum (L, O). D–I are higher-magnification images of the indicated boxed regions in A–C. M–O are higher-magnification images of the boxed region in J–L, respectively. Scale bars: A–C, G–I, 200 μm; D–F and J–L, 100 μm.

Hs2st is required by Emx1-lineage but not Zic4-lineage cells for CC development

Having established that both Zic4 and Emx1 lineages need to synthesize HS for normal CC development, we next investigated whether 2-O HS sulfation of the HS is required in either lineage. Hs2st is the sole enzyme capable of imparting 2-O HS sulfation onto HS, so to determine the cellular requirement for 2-O HS sulfation, we deleted Hs2st in the Zic4 or Emx1 lineages. Control Hs2st^+/+ genotypes (Hs2st^+/+;Emx1^CreER and Hs2st^+/+;Zic4^Cre) displayed neither CC agenesis nor midline astroglial disorganization; the control embryo shown in Figure 5 (A,D,G,J; D and G are reproduced from Fig. 3H,J) is of the Hs2st^+/+;Emx1^CreER genotype. The CC and the midline astroglial structures form normally in Hs2st^fl/fl;Zic4^Cre conditional mutants and the organization of L1⁺ axons and GFAP⁺ astroglia are indistinguishable from control embryos (6/6 embryos) (cf. Fig. 5B,A). The organization of GFP⁺ Zic4-lineage cells is the same in Hs2st^fl/fl;Zic4^Cre embryos as in Hs2st^+/+;Zic4^Cre embryos (cf. Figs. 5E,H and 3B,D) and IG Sox9⁺ cell counts confirm that the numbers of Sox9⁺ cells in the IG are not significantly different from control Hs2st^+/+ embryos (Fig. 5M, cf. blue and orange bars), indicating no cell-autonomous requirement for Hs2st in the Zic4-lineage Sox9⁺ IG astroglia. To exclude the possibility of a compensatory mechanism by which the Hs2st^fl/fl;Zic4^Cre IG is populated by Hs2st^+/+ cells from a different lineage, we performed Hs2st immunohistochemistry and confirmed that Hs2st expression is indeed absent from all cells in the IG (Fig. 5K, note this is an adjacent section from the same embryo to the one shown in H). In Hs2st^fl/fl;Emx1^CreER embryos, the CC fails to form in ∼50% of cases and embryos either had a severe phenotype (Fig. 5C, 5/9 embryos) or appeared completely unaffected (Fig. 5C′, 4/9 embryos). CC axons form Probst bundles on either side of the telencephalic midline, and the GFAP⁺ IG is expanded (asterisks in Fig. 5C). The anatomy and incomplete penetrance of the CC phenotype in Hs2st^fl/fl;Emx1^CreER embryos closely resemble constitutive null Hs2st^−/− embryos, indicating that Hs2st function within the Emx1 lineage is sufficient for normal CC development (Conway et al., 2011; Clegg et al., 2014). As in control Hs2st^+/+;Emx1^CreER embryos (Fig. 5D,G), the GFP and Sox9 signals did not overlap in the IG region of control or Hs2st^fl/fl;Emx1^CreER embryos (Fig. 5F, boxed area shown at higher magnification in I) and the Sox9⁺ cells in the IG of control embryos and the expanded IG of Hs2st^fl/fl;Emx1^CreER embryos were GFP⁻ (arrows in Fig. 5I indicate Sox9⁺;GFP⁻ cells). Counts of Sox9⁺ cells confirmed a significant increase in the IG of affected Hs2st^fl/fl;Emx1^CreER embryos compared with controls (Fig. 5M, cf. blue and purple bars). Immunostaining for Hs2st on adjacent sections confirmed that IG cells in Hs2st^fl/fl;Emx1^CreER embryos retain Hs2st protein expression (Fig. 5L). Because Sox9⁺ IG astroglia do not belong to the Emx1 lineage, their ectopic position in Hs2st^fl/fl;Emx1^CreER embryos despite retaining Hs2st function allows us to conclude a non-cell-autonomous requirement for Hs2st in the translocation of astroglial precursors to the IG.

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Figure 5.

Hs2st expression is required within Emx1-lineage cells but not Zic4-lineage cells for CC formation. A–C, Immunofluorescence for L1 and GFAP at E18.5. In control embryos, the U-shaped CC has formed and the IG can be observed above the CC (A). In Hs2st^fl/fl Zic4^cre embryos, the CC and IG form normally (B). In approximately half of Hs2st^fl/fl;Emx1^creER embryos, the CC fails to form, IG glia also extend ventrally (asterisks, C). In the remaining Hs2st^fl/fl;Emx1^creER embryos, the CC forms normally (C′). D–I, Immunofluorescence for Sox9 labels progenitor cells at the VZ and mature glia at the IG, GFP labels cells in which cre is active. D, G, In control (Hs2st^+/+;Emx1^creER) embryos, IG glia do not express GFP. E, H, In Hs2st^fl/fl;Zic4^cre embryos, IG glia do express GFP and adopt their normal position. F, I, In Hs2st^fl/fl;Emx1^creER embryos, GFP is expressed by cortical neurons and axons but not by abnormally positioned IG glia. J–L, Immunohistochemistry for Hs2st shows expression of Hs2st in the IG. J, In control embryos, punctate Hs2st staining can be seen within IG cells. In Hs2st^fl/fl Zic4^cre embryos, Hs2st is not expressed by IG glia (K). In Hs2st^fl/fl;Emx1^creER embryos, Hs2st is expressed by displaced glial cells (L). Hs2st immunohistochemistry in J–L was performed on adjacent tissue sections to those in D–I. M, Quantification of Sox9 expressing cell number at the IG in control (blue bar, n = 4 embryos, 2 Hs2st^+/+;Zic4^cre+ 2 Hs2st^+/+;Emx1^creER), affected Hs2st^fl/fl;Emx1^creER (orange bar, n = 4 embryos), and Hs2st^fl/fl;Zic4^cre (purple bar, n = 3 embryos). Sox9⁺ numbers are significantly increased compared with control in Hs2st^fl/fl;Emx1^creER embryos (*p < 0.05; F_(2,7) = 42.16, p = 0.00013, ANOVA, post hoc t tests: control vs Hs2st^fl/fl;Emx1^creER; t₍₄₎ = −8.08, p = 0.0013, t test; and control vs Hs2st^fl/fl;Zic4^cre; t₍₅₎ = 0.92, p = 0.40, t test). G–I are higher-magnification images of boxed regions in D–F, respectively. Insets in J–L are higher-magnification images of boxed region shown on each image. Scale bars: A–F, 200 μm; G–L, 50 μm.

The salient conclusions from these conditional mutagenesis experiments are that, although the Zic4-lineage astroglia do require Ext1 to form midline astroglial structures, they do not require Hs2st, strongly suggesting that, whereas these Zic4-lineage cells require HS on their cell surface to respond to translocation signals, there is no need for the HS to be 2-O sulfated. In contrast, Hs2st is absolutely required in the surrounding Emx1-lineage cells, indicating a non-cell-autonomous mechanism by which 2-O HS sulfation controls the transmission of translocation signals to the Zic4-lineage astroglial precursors.

Hs2st is not required cell autonomously by CC axons to navigate the midline

The conditional mutagenesis experiments showed that Hs2st has a non-cell-autonomous role in GW→IG somal translocation, but because Hs2st is expressed throughout the cerebral cortex, did not resolve whether there is an additional cell autonomous requirement in CC axon navigation. To solve this, we performed ex vivo transplantation experiments in which cerebral cortical tissue from transgenic mice ubiquitously expressing τGFP, which efficiently labels axons of τGFP⁺ cells, was transplanted into τGFP⁻ telencephalic slices containing the CC axon pathway and CSB structures (Pratt et al., 2000; Niquille et al., 2009). When WT E17.5 τGFP⁺ cortical explants are transplanted into age-matched τGFP⁻ WT cortical slices, τGFP⁺ axons extend across the telencephalic midline, forming the characteristic U shape of the CC and reaching the cortex of the opposite hemisphere (n = 3/3 cultures, arrows in Fig. 6A,D point to crossing axons). When Hs2st^−/− τGFP⁺ cortical explants are transplanted into τGFP⁻ WT slices, axons are able to cross the midline to reach the opposite hemisphere in a manner indistinguishable from that seen in the WT→WT transplants (n = 4/4 cultures, arrows in Fig. 6B,E point to crossing axons). In contrast, when τGFP⁺ WT cortical explants are transplanted into τGFP⁻ Hs2st^−/− slices, axons are unable to reach the opposite cortical hemisphere and instead remain within the cingulate cortex or invade the septum (n = 6/6 cultures, Fig. 6 C,F, arrowheads point to axons growing into the septum), resembling the in vivo CC phenotype observed in Hs2st^−/− embryos (Conway et al., 2011; Clegg et al., 2014). In all cultures, a few axons grew into the septum (arrowheads in Fig. 6D–F). Schematics summarizing these experiments are shown in Figure 6, G–I. These data show that 2-O HS sulfation is not required cell autonomously by CC projection neurons for axon guidance across the midline, strongly suggesting that disorganization of midline guidepost astroglial cells is the primary cause of the Hs2st^−/− CC agenesis phenotype.

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Figure 6.

Hs2st is not required by CC axons to cross the telencephalic midline. A, D, After homotypic transplantation of E17.5 cortical explants from GFP⁺ control tissue into the cortex of GFP⁻ control brain slices, GFP⁺ CC axons are able to project across the midline (arrows, D). B, E, After transplantation of GFP⁺ Hs2st^−/− cortical explants into GFP⁻ control brain slices, GFP⁺ CC axons are able to project across the midline (arrows, E). C, F, After transplantation of cortical explants from GFP⁺ control tissue into the cortex of GFP⁻ Hs2st^−/− brain slices, GFP⁺ CC axons are unable to project across the midline and invade the septum. Arrowheads indicate axons navigating into the septum in all conditions. G–I, Schematic of transplant experiments shown in A–C. D–F are higher-magnification images of the boxed region in A–C, respectively. Scale bars, 200 μm in all panels.

Abnormally high FGF/ERK signaling causes the Hs2st^−/− precocious astroglial translocation phenotype

We previously reported a correlation between hyperactive ERK signaling at the CSB and precocious somal translocation of astroglia to the midline in Hs2st^−/− embryos, but we did not formally establish that this stemmed from hyperactive FGF/ERK signaling (Clegg et al., 2014; Chan et al., 2017). To address this, we used an ex vivo assay in which coronal WT or Hs2st^−/− telencephalic slices incorporating the CSB were cultured on floating membranes for long enough to allow somal translocation to the midline and attempted to rescue the Hs2st^−/− phenotype by pharmacological abrogation of FGF/ERK signaling. WT or Hs2st^−/− E14.5 slices were cultured in the presence of the Fgfr1 inhibitor SU5402 dissolved in DMSO (FGFi treatment) to inhibit FGF/ERK signaling or in DMSO alone (untreated control) for 48 h (Fig. 7A). To aid subsequent identification of translocating cells, a subpopulation RGCs undergoing S-phase in the VZ at E14.5 were labeled just before culturing with a single pulse of BrdU. Immunohistochemistry for pErk (brown stain in Fig. 7B,D,F,H) confirms inhibition of FGF/ERK signaling in both FGFi treated WT and Hs2st^−/− cultures (Fig. 7D,H) compared with untreated cultures (Fig. 7B,F), showing that FGF signaling through Fgfr1 accounts for ERK phosphorylation in both genotypes. This demonstrates that ERK hyperactivation in Hs2st^−/− embryos does not stem from an FGF-independent mechanism for ERK activation (Clegg et al., 2014; Chan et al., 2017). After 48 h, some Sox9⁺ cells (red) had left the VZ and translocated to the midline in untreated WT cultures (arrow in Fig. 7C), with many more populating the midline in untreated Hs2st^−/− cultures (arrow in Fig. 7G), validating that our ex vivo assay replicates the in vivo Hs2st^−/− phenotype. Consistent with our hypothesis, FGFi treatment of both WT and Hs2st^−/− cultures resulted in a large decrease in Sox9⁺ cells reaching the midline (cf. Fig. 7E,I and C,G). We quantified glial translocation by counting the numbers of Sox9⁺ cells born in the VZ at E14.5 (Sox9⁺;BrdU⁺ cells, yellow; inset in Fig. 7C,E,G,I shows higher magnification) that had exited the VZ toward the midline (VZ demarcated by dotted line in Fig. 7C,E,G,I) after 2 d in culture. Counts of BrdU⁺;Sox9⁺ cells showed that glial translocation was significantly greater in Hs2st^−/− compared with WT cultures along the rostrocaudal axis (dark purple and green lines in Fig. 7J) and in both cases was almost completely suppressed by FGFi treatment (pale purple and green lines in Fig. 7J).

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Figure 7.

Hyperactive Fgf signaling causes precocious glia translocation in Hs2st^−/− CSB. A, Experimental outline of Hs2st^−/− phenotypic rescue experiment. Pregnant females were injected at E14.5 with a BrdU pulse and CSB slices collected after 1 h and cultured for 48 h. B–I, WT or Hs2st^−/− CSB slices were cultured in the presence of either SU5402 (FGFi) or DMSO (untreated vehicle control). B, D, F, H, pErk immunohistochemistry showing that FGFi treatment reduces Fgf/ERK signaling. (C,E,G,I) Immunofluorescence for BrdU and Sox9 in WT (C,E) and Hs2st^−/− (G,I) slices treated with FGFi (E,I) or untreated (C,G). Curved dotted line demarcates the basal edge of the VZ, arrows in C and G point to accumulations of BrdU/Sox9⁺ cells at the midline (arrow size corresponds to cell number), with higher magnification insets showing Sox9/BrdU⁺ (yellow) double-labeled cells in IG region. J, Quantification of Sox9/BrdU⁺ double-labeled cells in WT or Hs2st^−/− CSB slice cultures treated with FGFi or untreated (n = 3 embryos for each condition). For both genotypes, FGFi treatment significantly reduced the number of Sox9/BrdU⁺ cells that exit the VZ and moved toward the IG at one or more rostrocaudal position [significant differences due to FGFi treatment within each genotype indicated on graph as *p < 0.05, **p < 0.01; F_(3,32) = 31.00, p = 0.0000000014, two-way ANOVA, followed by t test with Sidak's correction for multiple comparisons at each positon along the rostrocaudal axis. WT FGFi vs WT untreated: position 1 (t₍₁₆₎ = 1.67, p = 0.24, t test); position 2 (t₍₁₆₎ = 2.37, p = 0.11, t test); position 3 (t₍₁₆₎ = 2.25, p = 0.15, t test); and position 4 (t₍₁₆₎ = 2.81, p = 0.050, t test). Hs2st^−/− FGFi vs Hs2st^−/− untreated: position 1 (t₍₁₆₎ = 2.38, p = 0.11, t test); position 2 (t₍₁₆₎ = 2.83, p = 0.048, t test); position 3 (t₍₁₆₎ = 3.05, p = 0.030, t test); and position 4 (t₍₁₆₎ = 4.60, p = 0.0012, t test]. Scale bars in B–I, 100 μm.

We conclude that the precocious glial translocation phenotype in Hs2st^−/− embryos is caused by hyperactive FGF/ERK signaling from E14.5 on. Together with our Hs2st conditional mutagenesis experiments demonstrating a non-cell-autonomous role for Hs2st in astroglial precursor translocation, we hypothesize that Hs2st normally suppresses the supply of FGF proteins to translocation competent astroglial precursors in the GW.

Hs2st suppresses Fgf17 protein levels

We next sought to identify an FGF protein that is targeted by Hs2st. Despite its well known role in CC development, Fgf8 protein levels are not significantly increased at the CSB of Hs2st^−/− embryos, forcing us to consider other FGFs (Clegg et al., 2014; Chan et al., 2017). A promising candidate is Fgf17, a member of the Fgf8 subfamily transcribed at the CSB in a similar pattern to Fgf8 (Cholfin and Rubenstein, 2008; Zhang X et al., 2012). Fgf17 is a canonical FGF that binds to HS, so it is potentially regulated via its interaction with HS and is known to play a role in patterning the telencephalon, although its role in CC development has not been fully characterized (Cholfin and Rubenstein, 2007; Hoch et al., 2015; Li and Kusche-Gullberg, 2016). We hypothesized that Hs2st normally suppresses Fgf17 protein and predicted that Fgf17 protein levels would be increased at the Hs2st^−/− CSB. We compared the expression of Fgf17 protein in the developing CSB of WT and Hs2st^−/− embryos at three developmental stages, E12.5, E14.5, and E16.5, spanning the interval of midline glial translocation. At E12.5, telencephalic Fgf17 protein is restricted to the CSB region with no obvious difference between WT and Hs2st^−/− (cf. Fig. 8A1,B1 and A2,B2). By E14.5, there is an expanded Fgf17 protein domain at the CSB of Hs2st^−/− embryos (cf. Fig. 8D1,E1 and D2,E2; *E2 marks the expanded Fgf17 protein domain). Quantification of Fgf17 immunofluorescence shows a significant ∼2-fold increase in Fgf17 protein levels in this region of Hs2st^−/− CSB (Fig. 8W, cf. blue and green bars). At E16.5, Fgf17 protein is much closer to detection threshold than at the earlier stages in both genotypes (Fig. 8G1,G2,H1,H2) although the increased protein spread in the mutant persists (* in Fig. 8H2), indicating that the Hs2st^−/− CSB is exposed to a prolonged overdose of Fgf17 protein spanning E14.5–E16.5. We next examined Fgf17 mRNA at the CSB to determine whether the increase in Fgf17 protein in Hs2st^−/− CSB was underpinned by altered Fgf17 gene expression. There was no evidence for this at E12.5 or E14.5, when the Fgf17 mRNA expression pattern remained similar between Hs2st^+/+ and Hs2st^−/− embryos (cf. Fig. 8C1,C2 and F1,F2); however, the expression domain of Fgf17 mRNA is increased in E16.5 Hs2st^−/− CSB (cf. Fig. 8I1and I2_, * in I2 marks expanded Fgf17 mRNA domain). This subsequent increase in Fgf17 mRNA in the E16.5 Hs2st^−/− CSB indicates that the Hs2st^−/− phenotype has a transcriptional component or that there are more cells expressing Fgf17 mRNA in the expanded Hs2st^−/− IG, although this cannot be the primary event because it is not apparent at E14.5, the stage at which we previously showed that precocious astroglial precursor translocation was well under way in Hs2st^−/− embryos (Clegg et al., 2014).

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Figure 8.

Expression of Fgf17 during CSB development in WT and Hs2st^−/− and Hs6st1^−/− embryos. A–C, Fgf17 protein (A,B) and mRNA (C) expression at the E12.5 CSB of WT and Hs2st^−/− embryos. Fgf17 protein is expressed across the CSB in both WT and Hs2st^−/− embryos, with no obvious change in intensity or domain of expression. Fgf17 mRNA expression overlaps well with the protein expression domain and is similar between WT and Hs2st^−/−. D–F, Fgf17 protein (D, E) and mRNA (F) expression at the E14.5 CSB of WT and Hs2st^−/− embryos. Fgf17 protein is expressed at low levels at the CSB of WT embryos. In Hs2st^−/− embryos, the protein expression domain expands across the CSB (asterisks in E2). Fgf17 mRNA is unchanged at the CSB between WT and Hs2st^−/− embryos. G–I, Fgf17 protein (G, H) and mRNA (I) expression at the E16.5 CSB of WT and Hs2st^−/− embryos. Fgf17 protein is expressed at low levels at the CSB of WT embryos (H1). In Hs2st^−/− embryos, the protein expression domain expands (asterisks, H2). There is a concurrent increase in Fgf17 mRNA (asterisk, I2). J–O, Fgf17 mRNA (J, K1– K3, L1–L3) and Fgf17 protein (M, N1–N3, O1–O3) expression (red) relative to GFP⁺ Zic4-lineage cells (indicated with white arrows) at the GW (K1–K3, N1–N3) and IG (L1–L3, O1–O3) of WT E14.5 embryos. P, E14.5 expression of Hs2st by LacZ staining. Hs2st is expressed most highly at the VZ, with decreasing expression toward the pial surface. Q–V, Fgf17 protein (Q, R) and mRNA (S) expression at the E14.5 CSB of WT and Hs6st1^−/− embryos. Fgf17 protein is expressed at low levels at the CSB of WT embryos (Q1, R1). In Hs6st1^−/− embryos, the protein expression domain is similar to WT (Q2, R2). Fgf17 mRNA expression is unchanged between WT and Hs6st1^−/− embryos (S1, S2). T–V, Fgf17 protein (T, U) and mRNA (V) expression at the E16.5 CSB of WT and Hs6st1^−/− embryos. Fgf17 protein is expressed at very low levels at the CSB of both WT (T1, U1) and Hs6st1^−/− (T2, U2) embryos. Fgf17 mRNA expression is unchanged between WT (V1) and Hs6st1^−/− (V2) embryos. W, Quantification of Fgf17 immunofluorescence signal at E14.5 CSB in WT (blue bar, n = 3 embryos), Hs2st^−/− (green bar, n = 3 embryos), and Hs6st1^−/− (purple bar, n = 3 embryos). Fgf17 protein level is significantly increased compared with WT in Hs2st^−/− embryos (*p < 0.05 on graph, F_(2,9) = 13.83, p = 0.0018, ANOVA, post hoc t tests: WT vs Hs2st^−/−, t₍₄₎ = −4.22, p = 0.014, t test; and WT vs Hs6st1^−/−, t₍₆₎ = −0.98, p = 0.36, t test). Boxed areas in A, D, G, Q, and T are shown at higher magnification in B, E, H, R, and U, respectively. Scale bars: A, D, G, Q, T, 200 μm; B, C, E, F, H, I, R, S, U, V, J, M, P, 100 μm; K, L, N, O, 10 μm.

Mosaic analysis (Fig. 5) indicated that Hs2st function in the Emx1 lineage negatively regulates a signal promoting GW→IG translocation of Zic4-lineage glial cells by a non-cell-autonomous mechanism and Fgf17 expression analysis (Fig. 8) makes Fgf17 a strong candidate for the signal. Based on this, we hypothesized that Fgf17 is expressed in cells surrounding the Zic4-lineage cells and performed detection of Fgf17 mRNA or protein in E14.5 WT embryos in which the Zic4 lineage is labeled GFP⁺. Fgf17 mRNA is expressed at the GW and the IG (Fig. 8J) and higher-power magnification shows that, in the VZ, GFP⁺ cells express little if any Fgf17 mRNA and, conversely, cells expressing the highest levels of Fgf17 mRNA are GFP⁻ (Fig. 8K1–K3, arrows indicate GFP⁺ cell location). This complementarity between Fgf17 mRNA-expressing and Zic4-lineage cells is preserved at the IG (Fig. 8L1–L3, arrows indicate GFP⁺ cell location). Fgf17 protein predominates at the IG (Fig. 8M) and higher-power magnification shows that, although Fgf17 protein is barely detectable at the GW (Fig. 8N1–N3), there are a number of much more highly Fgf17-expressing cells at the IG and these cells are GFP⁻, confirming that they do not belong to the Zic4 lineage (Fig. 8O1–O3, arrows indicate GFP⁺ cell location). Interestingly, although cells in the GW and IG express comparable levels of Fgf17 mRNA (cf. Fig. 8K1,L1), the expression of Fgf17 protein is much higher in the IG (cf. Fig. 8N1,O1), suggesting a posttranscriptional repression selectively at the GW. Our identification of Hs2st as a repressor of Fgf17 protein levels at this stage makes Hs2st a strong candidate; indeed, closer examination of Hs2st expression using the Hs2st-LacZ reporter shows that Hs2st is expressed in a GW^High-IG^Low pattern (Fig. 8P, also apparent in the Hs2st immunohistochemistry; Fig. 1B) complementary to the GW^Low-IG^High Fgf17 protein distribution. Together, these data bolster the idea that Hs2st acts to suppress Fgf17 protein supply to Zic4-lineage cells via a posttranscriptional mechanism.

We conclude that Hs2st primarily suppresses the level and spread of Fgf17 protein emanating from the Emx1 lineage in the CSB.

Hs6st1 does not affect Fgf17 protein levels

We next addressed whether the ability of 2-O HS sulfation to suppress Fgf17 protein levels in vivo represented a specific function of Hs2st or was redundant with other HSTs. We chose to examine Hs6st1, an HST that catalyzes 6-O HS sulfation, because we have previously shown that Hs6st1 (but not Hs2st) suppresses levels of the closely related Fgf8 protein at the CSB in vivo (Clegg et al., 2014; Chan et al., 2017). However, we were unable to detect increased expression of Fgf17 protein (cf. Fig. 8Q1,R1 and Q2,R2 and T1,U1 and T2,U2) or Fgf17 mRNA (cf. Fig. 8S1,S2 and V1,V2) in Hs6st1^−/− compared with WT CSB at either E14.5 or E16.5. Quantification of Fgf17 immunofluorescence shows unchanged Fgf17 protein levels in this region of Hs6st1^−/− CSB (Fig. 8W, cf. blue and purple bars). These data demonstrate that the negative relationship between Hs2st and Fgf17 is selective in vivo because it does not apply to Hs6st1.

Exogenously applied Fgf17 phenocopies the Hs2st^−/− astroglial translocation phenotype

Our data suggest that the Hs2st^−/− phenotype stems from abnormally high levels of Fgf17 protein at the CSB, causing FGF/ERK hyperactivation and precocious somal translocation to the IG. This requires that Hs2st^−/− CSB cells are competent to respond to Fgf17 protein and that application of ectopic Fgf17 triggers precocious glial translocation, neither of which has been previously established. We redeployed the CSB ex vivo culture assay (Fig. 7) with the modification that beads soaked in either recombinant Fgf17 protein (Fgf17 treatment) or in BSA (control) were implanted into coronal slices of CSB region on either side of the midline (Fig. 9A). WT or Hs2st^−/− slices implanted with Fgf17 and BSA beads were cultured for 2 h before processing for Fgf17 (green signal) and pErk (red signal) double immunofluorescence (Fig. 9B). In both WT and Hs2st^−/− cultures, Fgf17 protein was detectable adjacent to the edge of the bead (green signal) and this activated ERK phosphorylation in a similar pattern (red signal) with no obvious differences between WT and Hs2st^−/−, indicating that Hs2st^−/− CSB tissue is competent to respond to Fgf17 (Fig. 9B, top row). The lack of Fgf17 or pERK signal in the BSA control (Fig. 9B, bottom row) confirms Fgf17 antibody specificity and that pERK activation was specifically induced by exogenously applied Fgf17. We performed Sox9/BrdU analysis (exactly as described above for the FGFi experiments; Fig. 7) to assess the impact of experimentally introduced Fgf17 on astroglial translocation to the midline in WT CSB slices after 48 h in culture and the results were dramatic. The side with the Fgf17-bead showed many more Sox9⁺ (red) cells in the IG region (large arrow on right side of Fig. 9C) than the side with the BSA bead (smaller arrow on left side of Fig. 9C). Quantification of Sox9⁺;BrdU⁺ (yellow) cells (Fig. 9D shows higher magnification of IG region) confirmed a significant increase in astroglial translocation to the midline along the rostrocaudal axis on the side exposed to Fgf17 [Fig. 9E, cf. green (Fgf17) with black (control) lines]. An important function of IG glia is to secrete Slit2 and repulsively guide CC axons in the correct trajectory across the midline. At E16.5, Slit2 mRNA is normally expressed in the IG region and, in Hs2st^−/− embryos, the midline Slit2 expression domain is expanded (cf. Fig. 2C,E and D,F; Slit2 expression domain is bracketed). Experimentally introduced Fgf17 is sufficient to phenocopy this aspect of the Hs2st^−/− phenotype in our ex vivo assay because the side exposed to the Fgf17 bead has a much larger Slit2 domain than the BSA-treated side [cf. left (control) and right (Fgf17) bracketed areas in Fig. 9F], consistent with precocious translocation of excessive numbers of Slit2⁺ IG glia.

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Figure 9.

Fgf17-bead experiment. A, Experimental outline of Fgf17 protein bead experiment in WT embryos. Pregnant females were injected at E14.5 with a BrdU pulse and CSB slices collected. One Fgf17 and one BSA bead were added to each side of the midline. B, Fgf17 protein and pErk after 2 h in culture. In both WT and Hs2st^−/− CSB slices, Fgf17 and pErk are seen in tissue surrounding the Fgf17 bead. Staining for either is absent around the BSA bead (demarcated by dotted circle). C, Immunofluorescence for BrdU and Sox9 was performed on WT slices after 48 h in culture; curved dotted lines indicate the basal edge of the VZ and straight dotted line indicates the midline. D, Higher magnification of the arrowed regions in C. E, Quantification of Sox9⁺/BrdU⁺ double-labeled cells in WT CSB slice cultures with Fgf17 or BSA bead. The Fgf17 bead significantly increased the number of Sox9⁺/BrdU⁺ cells that exit the VZ and moved toward the IG (significant differences indicated on graph as **p < 0.05, ***p < 0.001) at the four caudal-most positions (n = 5 embryos; F_(1,48) = 65.63, p = 0.000000000155, two-way ANOVA, followed by t test with Sidak's correction for multiple comparisons for Fgf17-bead vs BSA-bead at each rostral-caudal position: position 1, t₍₄₈₎ = 1.45, p = 0.63, t test; position 2, t₍₄₈₎ = 2.36, p = 0.13, t test; position 3, t₍₄₈₎ = 3.65, p = 0.0039, t test; position 4, t₍₄₈₎ = 4.47, p = 0.0003, t test; position 5, t₍₄₈₎ = 4.03, p = 0.0012, t test; and position 6, t₍₄₈₎ = 3.89, p = 0.0018, t test). F, Slit2 expression in slices cultured with Fgf17 and BSA beads. Scale bars, 100 μm.

We conclude that Hs2st^−/− CSB tissue is competent to respond to Fgf17 protein and abnormally high levels of Fgf17 protein are sufficient to phenocopy the Hs2st^−/− astroglial translocation phenotype consistent with the model presented in Figure 10.

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Figure 10.

Model. Hs2st expressed in Emx1-lineage cells catalyzes 2-O HS sulfation (2-O HS) that in turn suppresses the levels of Fgf17 protein, but not Fgf8 protein, by an unknown mechanism at the CSB. Zic4 lineage astroglial precursors respond to Fgf8 and Fgf17 protein by activating FGF/ERK signaling and translocating (black arrows) to the midline. This generates appropriate positioning of Slit2⁺ astroglia to guide CC axons across the midline. Loss of 2-O HS from the Emx1 lineage selectively desuppresses Fgf17 protein levels leaving Fgf8 protein unaffected. This causes hyperactive FGF/ERK signaling and more Zic4 lineage astroglial precursors translocate than normal with consequent blocking of CC axon midline crossing by the ectopic midline Slit2⁺ astroglia. Zic4 lineage astroglial precursor cells do not need to express 2-O HS to respond to FGF signaling proteins and translocate to the midline.

Hs2st selectively facilitates physical interaction between Fgf17 protein and HS

Our in vivo data show that Hs2st suppresses the levels of Fgf17 protein and that this represents a selective interaction between Hs2st-mediated 2-O HS sulfation and Fgf17 protein levels in vivo because Hs2st does not suppress the levels of the closely related Fgf8 protein, and Hs6st1, which catalyzes 6-O HS sulfation, does not suppress Fgf17 protein levels (Clegg et al., 2014; Chan et al., 2017) (Fig. 8). However, these in vivo experiments do not resolve whether differential sulfation has a correspondingly direct selective effect on the physical interaction between HS and Fgf17. To test the hypothesis that Hs2st has a selective effect on the binding of Fgf17 protein to HS molecules, we turned to a biochemical assay, the LACE assay, which probes the physical interaction between HS and FGF proteins by quantifying the ability of endogenous HS in tissue sections to form Fgf:Fgfr:HS complexes with exogenously added Fgf protein and Fgfr ectodomain fused to an Fc tag for immunofluorescent detection (Allen et al., 2001; Chan et al., 2015). We used the Fgf17:Fgfr1 LACE assay to compare the binding of Fgf17 protein to HS in WT, Hs2st^−/−, and Hs6st1^−/− CSB tissue at E14.5 and E16.5 to test the hypothesis that Fgf17:HS physical interaction is selectively sensitive to loss of 2-O HS sulfation in Hs2st^−/− tissue (Fig. 11A–J,O). We used the Fgf8:Fgfr3 LACE assay to compare the binding of Fgf8 protein to HS in WT and Hs2st^−/− CSB tissue at E14.5 to test the hypothesis that the Fgf8:HS physical interaction is insensitive to loss of 2-O HS sulfation in Hs2st^−/− tissue (Fig. 11K–N,P).

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Figure 11.

LACE assay for FGF:HS interactions. A–J, O, FGFR1/FGF17 LACE experiments on (A–E) E14.5 and (F–J) E16.5 telencephalic coronal sections through the CSB. A, F, WT. B, G, Hs2st^−/−. C, H, Hs6st1^−/−. D, I, WT sections pretreated with heparinitase to digest HS. E, J, WT sections with FGF17 omitted from the LACE assay. O, Quantification of FGF17/FGFR1 LACE signal in WT (blue bar, n = 9 embryos), Hs2st^−/− (green bar, n = 5 embryos), and Hs6st1^−/− (purple bar, n = 4 embryos), showing a significant decrease (*p < 0.05 on graph) in Hs2st^−/− embryos F_(2,15) = 8.62, p = 0.0032, ANOVA, followed by post hoc t test: WT vs Hs2st^−/−, t₍₉₎ = 6.11, p = 0.014, t test; WT vs Hs6st1^−/−, t₍₅₎ = 0.63, p = 0.56, t test). K–N, P, FGFR3/FGF8 LACE experiments on E14.5 telencephalic coronal sections through the CSB. K, WT. L, Hs2st^−/−. M, WT sections pretreated with heparinitase to digest HS. N, WT sections with FGF8 omitted from the LACE assay. P, Quantification of FGF8/FGFR3 LACE signal in WT (blue bar, n = 3 embryos) and Hs2st^−/− (green bar, n = 3 embryos) showing no significant difference (t₍₃₎ = 0.29, p = 0.76, t test). Numbers of embryos of each genotype analyzed indicated under bars. Q, Summary diagram. FGFR1/FGF17/HS complex formation is equally supported by WT and Hs6st1^−/− HS but less so by Hs2st^−/− HS, whereas FGFR3/FGF8/HS complex formation is equally supported by WT and Hs2st^−/− HS, showing that the FGF17:HS physical molecular interaction is selectively dependent on 2-O HS sulfation. A′–N′, Higher magnification showing the CSB region boxed in A–N (note that the DAPI channel is not shown in the higher-magnification images to improve visualization of the LACE signal). Scale bars, 200 μm.

In both E14.5 and E16.5 WT tissue, the Fgf17:Fgfr1 and Fgf8:Fgfr3 LACE assays produced a strong LACE signal (Fig. 11A,F,K, with higher magnification of boxed areas enclosing CSB region shown in A′,F′,K′). Control experiments show this LACE signal was drastically reduced by pretreating the tissue with heparitinase to digest HS (Fig. 11D,I,M, with higher magnification of CSB in D′,I′,M′) or omitting Fgf17 or Fgf8 protein from the assay (Fig. 11E,J,N, with higher magnification of CSB in E′,J′,N′). Together, these controls confirm that the LACE signal provides a specific readout of the interaction between each FGF protein and HS molecules. To determine the effect of differential sulfation on the physical interaction between HS and Fgf8 or Fgf17, we investigated how the LACE signal was affected when the assay was performed on Hs2st^−/− and Hs6st1^−/− tissue. As predicted by our hypothesis, the binding of Fgf17 to HS is selectively sensitive to 2-O HS sulfation because we found that the Fgf17:Fgfr1 LACE signal was much weaker than WT in Hs2st^−/− tissue (cf. B,B′,G,G′ and A,A′,F,F′), but similar to WT in Hs6st1^−/− tissue (cf. C,C′,H,H′ and A,A′,F,F′). Quantification of Fgf17:Fgfr1 LACE signal intensity in Figure 11O shows a significant ∼4-fold reduction in Hs2st^−/− (green bar) compared with WT (blue bar), but no significant difference from WT in Hs6st1^−/− (purple bar). As predicted by our hypothesis that the binding of Fgf8 to HS is not sensitive to 2-O HS sulfation, we found that there was no difference in the Fgf8:Fgfr3 LACE signal between WT and Hs2st^−/− tissue (cf. Fig. 11K,K′ and L,L′). Quantification of Fgf8:Fgfr3 LACE signal intensity in Figure 11P shows no significant difference between Hs2st^−/− (green bar) compared with WT (blue bar). These LACE results are summarized schematically in Figure 11Q, which shows that, of the five HST genotype and FGF ligand permutations tested, only the Fgf17:HS physical interaction was sensitive to the Hs2st genotype, as predicted by the hypothesis that 2-O HS sulfation has a specific effect on the ability of HS to bind Fgf17.