Heparan sulfate sugar modifications mediate the functions of slits and other factors needed for mouse forebrain commissure development

Abstract

Heparan sulfate proteoglycans are cell surface and secretory proteins that modulate intercellular signaling pathways including Slit/Robo and FGF/FGFR. The heparan sulfate sugar moieties on HSPGs are subject to extensive postsynthetic modification, generating enormous molecular complexity that has been postulated to provide increased diversity in the ability of individual cells to respond to specific signaling molecules. This diversity could help explain how a relatively small number of axon guidance molecules are able to instruct the extremely complex connectivity of the mammalian brain. Consistent with this hypothesis, we previously showed that mutant mice lacking the heparan sulfotransferases (Hsts) Hs2st or Hs6st1 display major axon guidance defects at the developing optic chiasm. Here we further explore the role of these Hsts at the optic chiasm and investigate their function in corpus callosum development. Each Hst is expressed in a distinct pattern and each mutant displays a specific spectrum of axon guidance defects. Particular Hs2st(-/-) and Hs6st1(-/-) phenotypes closely match those of Slit1(-/-) and Slit2(-/-) embryos respectively, suggesting possible functional relationships. To test functional interactions between Hs2st or Hs6st1 and Slits we examined optic chiasm and corpus callosum phenotypes in a panel of genotypes where Hs2st or Hs6st1 and Slit1 or Slit2 function were simultaneously reduced or absent. We find examples of Hs2st and Hs6st1 having epistatic, synergistic, and antagonistic genetic relationships with Slit1 and/or Slit2 depending on the context. At the corpus callosum we find that Hs6st1 has Slit-independent functions and our data indicate additional roles in FGF signaling.

Figure 1.

Slit and Hst;Slit double mutant inter-retinal innervation phenotypes at E15.5. A–F, DiI labeling of RGCs projecting to the contralateral retina in wild-type (A), Slit1^−/− (B), Slit2^−/− (C, D), Hs2st^−/−;Slit1^−/− (E), and Hs6st1^−/−;Slit2^−/− (F) embryos. Although RGCs project to the contralateral retina in all genotypes, more are retrogradely labeled in Slit2^−/− (C) (D is a magnification of box in C showing retrogradely labeled RGC bodies) and Hs6st1^−/−;Slit2^−/− embryos (F). G, Quantification of numbers of RGCs retrogradely labeled by DiI injection into the contralateral retina in wild-type, Hs2st^−/−, Slit1^+/−, Slit1^−/−, Hs2st^−/−;Slit1^−/−, Hs6st1^−/−, Slit2^+/−, Slit2^−/−, and Hs6st1^−/−;Slit2^−/− embryos. Hs6st1^−/− (sixth bar), Slit2^+/− (seventh bar), Slit2^−/− (eighth bar), Hs6st1^−/−;Slit2^−/− (ninth bar), and Slit1^−/−;Slit2^−/− (10th bar) values are significantly greater than wild-type. Slit2^−/− and Hs6st1^−/− values are not significantly different from each other, both are significantly different from the Hs6st1^−/−;Slit2^−/− genotype, and there is no significant difference between Hs6st1^−/−;Slit2^−/− and Slit1^−/−;Slit2^−/− genotype. Kruskal–Wallis one-way ANOVA on ranks between all groups p < 0.001. p Values for statistically significant differences (p < 0.05) are in bold. Numbers above bars indicate p values for pairwise comparison of ranks for each genotype to wild-type using Dunn's method. p Values for pairwise comparisons between other genotypes are shown above horizontal lines bracketing the genotypes. Error bars are ±SEM. Numbers of embryos of each genotype analyzed are indicated at the very bottom. l, Lens; r, retina; onh, optic nerve head. All sections horizontal with caudal at top. Scale bars: A–C, E–F, 200 μm; D, 100 μm.

Figure 2.

Ectopic retinal ganglion cell axon growth through the preoptic area in Hs2st^−/− embryos. A–F, DiI injection into the retina labeling RGC axons at the optic chiasm in E15.5 embryos. DiI images are presented as grayscale negatives for clarity. B, E, Hs2st^−/− embryos RGC axons escape the normal chiasm region and grow ectopically across the preoptic area (E, green arrow) as well as dorsally up the midline (B, black arrow). This ectopic axon growth does not occur in wild-type (A, D) or Hs6st1^−/− (C, F) embryos. G–I, Merges of A and D (G), B and E (H), and C and F (I), with * marking side of DiI application. J–O, RGC axons stained brown using neurofilament immunohistochemistry. J–O, E15.5. Wild-type embryo (J, M), Hs2st^−/− embryo (K, N), Hs6st1^−/− embryo (L, O). An ectopic branch of the optic nerve enters the preoptic area in Hs2st^−/− embryos (K, N, green arrows) which is absent in the other genotypes. P–R, Diagrams summarizing RGC axon navigation at the chiasm of wild-type, Hs2st^−/−, and Hs6st1^−/− embryos; green arrows, preoptic area tract; black arrows, midline wandering; red arrow (R), increased inter-retinal innervation previously described (Pratt et al., 2006). on, Optic nerve; oc, optic chiasm; ot, optic tract; r, retina; l, lens; poa, preoptic area; M, midline. All sections horizontal with caudal at top. Scale bars: A–L, 500 μm; M–O, 250 μm.

Figure 3.

Hst;Slit double mutant optic chiasm phenotypes at E15.5. A–H, Consecutive horizontal sections following unilateral DiI injection into the retina labeling RGC axons at the optic chiasm in E15.5 embryos. DiI images are presented as grayscale negatives for clarity. A–H, Wild-type (C, F), Hs2st^−/−;Slit1^−/− (A, D, G), and Hs6st1^−/−;Slit2^−/− (B, E, H) embryos. Hs2st^−/−; Slit1^−/− embryos exhibit ectopic RGC axon growth dorsally and caudally (A, D, black arrows) up the midline as well as ectopic growth into the preoptic area (D, green arrow). B, E, H, Hs6st1^−/−;Slit2^−/− embryos exhibit ectopic dorsal and caudal growth up the midline (E, H, black arrows), ectopic growth into the preoptic area (E, green arrow) and an ectopic tract growing laterally from the optic chiasm (B, blue arrow). I–K, Merges of C and F (I); A, D, and G (J); and B, E, and H (K), with * marking side of DiI application. L—N, Coronal sections through the optic chiasm following unilateral DiI labeling of the retina. N, Hs6st1^−/−;Slit2^−/− embryos an ectopic axon tract grows over the surface of the ventral telencephalon (blue arrow), which is not seen in wild-type (L) or Hs2st^−/−;Slit1^−/− (M) embryos. O–Q, Diagrams summarizing the phenotypes; green arrows, preoptic area tract; black arrows, midline wandering; red arrows, increased inter-retinal innervation. on, Optic nerve; oc, optic chiasm; ot, optic tract; r, retina; l, lens; poa, preoptic area; M, midline. Scale bars, 500 μm.

Figure 4.

Expression of Slit1, Slit2, Robo1, and Robo2 mRNA in the retina and optic chiasm of wild-type, Hs2st^−/−, and Hs6st1^−/− embryos at E14.5. A–P, Slit1 (A–D), Slit2 (E–H), Robo1 (I–L), Robo2 (M–P) . A–C, E–G, I–K, M–O, In situ hybridization on horizontal sections. D, H, L, P, qRT-PCR on RNA extracted from optic chiasm (D, H) or eye (L, P) with mRNA levels normalized to GAPDH. For each transcript, normalized mRNA levels were compared between genotypes using Kruskal–Wallis one-way ANOVA on ranks. In situ hybridization showed indistinguishable expression patterns of Robo1/2 and Slit1/2 mRNAs between genotypes in the retina and at the optic chiasm. qRT-PCR did not reveal any significant differences in the levels of Robo and Slit mRNAs between wild-type, Hs2st^−/−, or Hs6st1^−/− embryos except for Slit1 mRNA which was significantly reduced at the optic chiasm of Hs6st1^−/− embryos (D). r, Retina; oc, optic chiasm; poa, preoptic area; l, lens. All sections horizontal with caudal at top. Scale bars: A–G, 500 μm; I–O, 250 μm.

Figure 5.

Hs2st^−/− and Hs6st1^−/− corpus callosum axon navigation phenotypes. A–I, Callosal axons were labeled by injecting DiI (red) and DiA (green) into the cerebral cortices of E18.5 mouse brains. A, D, G, In wild-type embryos, callosal axons cross the midline and form the characteristic “U” shape of the corpus callosum. At E18.5, a large number of callosal axons cross the midline along the rostral to caudal axis. Similar DiI/DiA injections in Hs2st^−/− (B, E, H) and Hs6st1^−/− (C, F, I) embryos reveal distinct corpus callosum axon navigation phenotypes. B, E, H, In Hs2st^−/− embryos large knotted Probst-like axon bundles develop on either side of the midline and the callosal axons completely fail to cross the midline. C, F, I, In Hs6st1^−/− embryos large knotted Probst-like axon bundles develop on either side of the midline and the callosal axons completely fail to cross the midline. More axons grow ectopically into the septum (white arrows) than in other genotypes. J–L, Schematic representations of corpus callosum phenotypes observed in wild-type (J), Hs2st^−/− (K), and Hs6st1^−/− (L) embryos. P, Probst-like knotted axons; gray arrow, sepatal axons; purple arrow, callosal axons crossing the midline. GFAP-expressing glia (see Fig. 6) are represented by green ovals. ctx, Cerebral cortex; cc, corpus callsoum; s, septum; P, knotted Probst-like axon bundle; s, septum; M, midline; vtel, ventral telencephalon. All sections coronal with dorsal at top. Scale bars, 200 μm.

Figure 6.

Development of telencephalic midline neuronal and glial structures critical for corpus callosum development in Hs2st^−/− and Hs6st1^−/− embryos. A–F, Tbr1 (green) is a marker for neurons that populate the IG and the SCS. A–C, E16.5. A, In wild-type embryos, Tbr1-expressing cells are located in the cortex and into the IG region (dotted box). This pattern is not altered in Hs2st^−/− (B) or Hs6st1^−/− (C) embryos. D–F, E18.5. D, In wild-type embryos Tbr1-expressing cells form a continuous sling at the ventral border of the corpus callosum called the SCS. Densely packed cells expressing high levels of Tbr1 mark the IG region (enclosed by dotted box) with less dense labeling in the cerebral cortex. E, Although Hs2st^−/− embryos have cortical and IG (dotted box) staining similar to wild-type embryos, the SCS pattern is different with a gap (white arrow) devoid of Tbr1-positive cells at the midline. F, In Hs6st1^−/− embryos densely packed Tbr1-expressing cells mark the IG (dotted box) but the Tbr1-expressing cells do not span the midline and there is a gap in the sling (white arrow). G–L, P–R, Immunohistochemistry for GFAP labels glia (P–R) are double labeled with the axonal marker L1 (red). G–I, E16.5. G, In wild types, glia are arranged in the GW in the ventricular zone of the corticoseptal boundary (yellow arrow). H, In Hs2st^−/− embryos the GFAP pattern is comparable to wild types at the GW (yellow arrow). I, Hs6st1^−/− embryos have a more severe phenotype with very few glia found in the GW at this stage (yellow arrow, glial fascicle). J–L, By E17.5 there are no longer gross differences in the GW (yellow arrows) but glial organization at the midline differs dramatically between genotypes. J, In wild-type embryos, glia have populated the IG above the corpus callosum. K, In Hs2st^−/− embryos, glia are present in the IG (dotted box) and there are a few ectopic glia in the path of corpus callosum axons (blue arrow). L, In Hs6st1^−/− embryos there are no glia populating the region of the IG (dotted box) and many ectopic glia are seen along the midline (blue arrow) and medial to the GW (black arrow). The cerebral hemispheres have become separated with the gap marked by an asterisk. M–O, Quantification of GFAP staining at the GW in wild-type, Hs2st^−/−, and Hs6st1^−/− embryos. M, Diagram illustrating the measurement of the length of the GW taken from the dorsal-most to the ventral-most GFAP stained glial process as described in Results. N, At E16.5 there are significant differences between all genotypes and the GW is by far the smallest in Hs6st1^−/− embryos. O, At E17.5, there is no significant difference between genotypes. Error bars are ±SEM with the number of embryos analyzed shown below each bar. Numbers above each bar indicate Student's t test p values for comparison to wild type or bracketed for comparison between mutants. Significant differences (p < 0.05) in bold. P–S, At E18.5 glia occupy the IG in wild-type (P, Q) and Hs2st^−/− (R) embryos but absent from the IG in Hs6st1^−/− (S) embryos (dashed boxes in Q–S enclose IG). More glia are seen at the midline in Hs2st^−/− embryos than in wild types (R, blue arrow) and in Hs6st1^−/− embryos large numbers of ectopic glia are found both at the midline (S, blue arrow) and forming ectopic strands intermingled with the axons (S, white arrow). s, Septum; vz, ventricular zone; ctx, cerebral cortex. All sections coronal with dorsal at the top. Scale bars: A–L, P, 200 μm; Q–S, 100 μm.

Figure 7.

Expression of Hs2st and Hs6st1 in the formation of the corpus callosum. Hs2st and Hs6st1 expression analysis using LacZ and hplap gene-trap reporter transgenes. A–M, Hs2st^+/− (A, C, G, J), Hs2st^−/− (D, K), Hs6st1^+/− (B, E, H, I, I', L), and Hs6st1^−/− (F, M) embryos at E16.5 (A–F) and E18.5 (G–M). A–H, J–M, LacZ histochemistry (blue). I, I', Hplap histochemistry (purple). At E16.5 both Hs2st (A, C) and Hs6st1 (B, E) are expressed widely in the telencephalon including the cerebral cortex, ventral telencephalon, and septum with variations in staining intensity across these structures. (D) Hs2st^−/− embryos show similar GW staining to (C) Hs2st^+/− embryos. F, Hs6st1^−/− embryos have similar patterns of expression to (E) Hs6st1^+/− embryos. G–M, At E18.5 LacZ expression in Hs2st^+/− (G) and Hs6st1^+/− (H) embryos are in distinct laminar patterns in the cerebral cortex. G, H, Red boxes, Regions shown at higher magnification in J and L. I, Weak hplap staining of corpus callosum axons in coronal section from Hs6st1^+/− embryo. I', Strong hplap staining of the anterior commissure crossing the midline at a more ventral location in the same section. J, Hs2st^+/− LacZ expression is high in the glial wedge and in the IG (red arrow) and the SCS (yellow arrows). K, The IG shows high LacZ expression in Hs2st^−/−, however unlike the Hs2st^+/− (J), there is high LacZ expression at the midline below the IG. L, Hs6st1^+/− LacZ is expressed strongly dorsal to the corpus callosum in the IG (red arrow). M, Hs6st1^−/− embryos have ectopically located LacZ cells intermingled with the corpus callosum axon bundles (dashed red arrow) and no apparent LacZ expression in the IG. ctx, Cerebral cortex; s, septum, vtel, ventral telencephalon; P, Probst-like bundle; cc, corpus callosum. All sections are coronal with dorsal at the top. Scale bars: A–B, G–H, 500 μm; C–F, I–M, 500 μm.

Figure 8.

Hs6st1 and Slit2 act in synergy to keep cortical axons out of the septum. A–F, Axon labeling following DiI and DiA injection into opposite cerebral cortices (ctx) of wild-type (A–C) and Hs6st1^+/−;Slit2^+/− (D–F) compound heterozygote E18.5 embryos. The overall structure of the corpus callosum is not affected (compare A to D), with large numbers of axons crossing the midline in both genotypes (compare B to E). C, F, Higher magnifications of the septal area demarcated by dotted box in A and D, showing more septal axons (white arrows) in Hs6st1^+/−;Slit2^+/− embryos (F) compared with wild-type. G, H, A semiquantitative analysis the of Slit2^+/−, Hs2st^+/−, and Hs6st1^+/− heterozygous and Hs6st1^+/−;Slit2^+/− Hs2st^+/−;Slit2^+/− compound heterozygous septal axon phenotypes. Consecutive sections though the corpus callosum region of each embryo were each scored blind to genotype according the following criteria: 0, no axons; 1, <10 axons; 2, >10 axons; 3, too many axons to count (∼100 or more axons). Graphs show means of septal phenotype scores for each genotype plotted against their postion on the rostro-caudal axis (1, most caudal to 7, most rostral). Numbers in square brackets are averages across all sections for each genotype. G, Hs6st1^+/−;Slit2^+/− embryos (pale blue) consistently scores higher than all other genotypes indicating a synergistic genetic interaction between Hs6st1 and Slit2 in preventing axons from transiting between the cerebral cortex and the septum. H, There is no clear difference between Hs2st^+/−;Slit2^+/− embryos and other genotypes providing no evidence of genetic interaction. Numbers of embryos scored: wild-type and Hs6st1^+/−, n = 4; all other genotypes, n = 3. All sections coronal with dorsal at the top. Scale bars, 200 μm.

Figure 9.

The complete failure of callosal axons to cross the midline in Hs6st1^−/− embryos is rescued in Hs6st1^−/−;Slit2^−/− embryos. A–E, Axon labeling following DiI and DiA injection into opposite cerebral cortices of Hs6st1^−/−;Slit2^−/− compound homozygote E18.5 embryos. A–C, Rostral-caudal series of section showing that although Probst-like axon whorls flank the midline, and many axons grow into the septum, large numbers of callosal axons are able to cross into the contralateral cerebral cortex (red arrows) with higher magnification of crossing axons shown in D. E, A higher magnification showing large numbers of axons in the septum (F) L1/GFAP immunohistochemistry of Hs6st1^−/−;Slit2^−/− embryo. Large numbers of ectopically positioned glia occupy the midline and callosal axons have been able to cross to form a corpus callosum (cc). G, Diagram illustrating the Hs6st1^−/−;Slit2^−/− corpus callosum phenotype. Gray arrow, ectopic growth into the septum; P, Probst-like knotted axons; *, severe midline glial defect; purple arrow, callosal axons crossing the midline. All sections coronal with dorsal at the top. Scale bars, 200 μm.

Figure 10.

Hs6st1 and FGF signaling in the corticoseptal region of E14.5 embryos. A, B, In situ hybridization for Sprouty2, which is transcriptionally upregulated in response to FGF signaling. A, Wild-type embryo. B, Hs6st1^−/− embryo. In both genotypes Sprouty2 is expressed in the telencephalic ventricular zone with particularly high expression at the corticoseptal boundary (A, B, red arrows) and no obvious difference in the expression pattern between genotypes. C, qRT-PCR for Sprouty2 mRNA (levels normalized to GAPDH) in RNA extracted from the E14.5 septum, corticoseptal boundary, and medial cortex. Sprouty2 levels are reduced by ∼25% in Hs6st1^−/− embryos indicating a reduction in FGF signaling. vtel, Ventral telencephalon; s, septum; ctx, cerebral cortex. Scale bars, 500 μm.

Figure 11.

Summary and comparison of optic chiasm and corpus callosum phenotypes. A–H, The optic chiasm. Diagrams show RGC axons navigating through the optic chiasm in the genotypes indicated at E15.5. Phenotypes: green arrow, ectopic growth though the poa; black arrow, midline wandering; red arrow, growth into the opposite eye; blue arrow, ectopic tract growing over the ventral telencephalon. A, Wild-type. B–E, Hst and Slit single mutant phenotypes (data in Figs. 1, 3) (Plump et al., 2002; Pratt et al., 2006). F, Slit1^−/−;Slit2^−/− diagram based on data from Plump et al. (2002), Thompson et al. (2006), and Plachez et al. (2008). G, H, Hst and Slit double mutant phenotypes (data in Figs. 2, 3). I–N, The corpus callosum. Diagrams show axons navigating through the corpus callosum and the distribution of GFAP-expressing glia (green ovals) in the GW, IG, and MZ in the genotypes indicated at E18.5. Phenotypes: gray arrow, ectopic growth into the septum; P, Probst-like knotted axons; *, severe midline glial defect; purple arrow, callosal axons crossing the midline. I, Wild-type. J, K, Hst single mutants (data in Figs. 5, 6). L–M, Slit/Robo and FGFR mutants. L, Slit2^−/−, Robo1^−/−, and Robo1^−/−;Robo2^−/− (Bagri et al., 2002; Andrews et al., 2006; López-Bendito et al., 2007). Foxg1^Cre;FGFR1^Fl/Fl conditional knockout of FGFR1 throughout the developing telencephalon using Foxg1^Cre in which midline glia are absent (Tole et al., 2006) (M) and GFAP^Cre;FGFR1^Fl/Fl is a more restricted conditional knockout of FGFR1 in glia using GFAP^Cre where glial distribution is disrupted (Smith et al., 2006) (N). O, Hs6st1^−/−;Slit2^−/− embryos in which callosal axons are able to cross the midline, in contrast to the Hs6st1^−/− phenotype (compare with K) where axons do not cross, and despite a major midline glial disorganization phenotype. r, Retina; l, lens; on, optic nerve; oc, optic chiasm; ot, optic tract; poa, preoptic area; ctx, cerebral cortex; vtel, ventral telencephalon; s, septum; P, Probst-like knotted axons.