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. 2014 Oct;243(10):1310-6.
doi: 10.1002/dvdy.24142. Epub 2014 May 6.

Role of heparan sulfate proteoglycans in optic disc and stalk morphogenesis

Zhigang Cai  1 , Kay Grobe, Xin Zhang
Affiliations

Role of heparan sulfate proteoglycans in optic disc and stalk morphogenesis

Zhigang Cai et al. Dev Dyn. 2014 Oct.

Abstract

Background: Heparan sulfate proteoglycans (HSPG) are important for embryonic development by means of the regulation of gradient formation and signaling of multiple growth factors and morphogens. Previous studies have shown that Bmp/Shh/Fgf signaling are required for the regionalization of the optic vesicle (OV) and for the closure of the optic fissure (OF), the disturbance of which underlie ocular anomalies such as microphthalmia, coloboma, and optic nerve hypoplasia.

Results: To study HSPG-dependent coordination of these signaling pathways during mammalian visual system development, we have generated a series of OV-specific mutations in the heparan sulfate (HS) N-sulfotransferase genes (Ndst1 and Ndst2) and HS O-sulfotransferase genes (Hs2st, Hs6st1, and Hs6st2) in mice. Of interest, the resulting HS undersulfation still allowed for normal retinal neurogenesis and optic fissure closure, but led to defective optic disc and stalk development. The adult mutant animals further developed optic nerve aplasia/hypoplasia and displayed retinal degeneration. We observed that MAPK/ERK signaling was down-regulated in Ndst mutants, and consistent with this, HS-related optic nerve morphogenesis defects in mutant mice could partially be rescued by constitutive Kras activation.

Conclusions: These results suggest that HSPGs, depending on their HS sulfation pattern, regulate multiple signaling pathways in optic disc and stalk morphogenesis.

Keywords: Fgf; HSPG; Ndst; optic disc; optic stalk; sulfotransferase.

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Figures

Figure 1
Figure 1. Comparable ocular defects in Six3-Cre; Ndst1flox/flox; Ndst2KO/KO and Six3-Cre; Hs6st1flox/flox; Hs6st2KO/KO; Hs2stflox/flox mice
(A–H) Conditional Ndst1/Ndst2 mutants displayed normal closure of the optic cup in the front (A–D), but optic nerve hypoplasia or aplasia at the back side of the eye (E and F, arrow). Retinal thinning was evident at adult stages (G and H). (I–P) Hs2st/Hs6st1/Hs6st2 conditional mutants also showed hypoplastic optic nerves and degenerating retinae.
Figure 2
Figure 2. Molecular markers for earlier neural retina development in Ndst mutants
(A–J) The NR marker Chx10, RPE markers Mitf and Dct1, and ciliary margin marker Otx1 are expressed normally in Ndst1/Ndst2 conditional mutants. (K–N) Sagittal sections of Ndst1/Ndst2 mutants showed that the optic cup is fused at the ventral side (arrows). (O–P) Transvers sections showed that Pax2 were reduced in the optic disc region (arrows). (Q–T) Sox2 and Brn3b expression indicates unaffected differentiation of retinal ganglion cells in conditional Ndst1/Ndst2 mutants. (U–X) No significant differences in the expression of cell proliferation markers Cyclin D1 and Ki-67 were observed in eyes of E13 Ndst1/Ndst2 conditional mutants.
Figure 3
Figure 3. Disrupted optic disc and stalk development in Ndst and Hs6st/2st conditional mutant mice
(A–D″) The optic disc markers Netrin-1 and Pax2 were down regulated in E14 Ndst1/Ndst2 mutants. Arrows indicate Netrin-1 and Pax2-expressing optic discs. (E–N) Abnormal development of optic stalks and nerves was observed in both E14 Ndst and Hs6st/2st mutants, as indicated by altered expression of NF-165 and Pax2. Arrowheads denote the misrouting of retinal ganglion cell axons. Optic stalks were marked by circling the cells surrounding the NF-165-expressing neural fibers in dashed white lines (L and N).
Figure 4
Figure 4. Ligand and carbohydrate engagement (LACE) analysis of disrupted HS-dependent Fgf-Fgfr assembly in Ndst mutants
(A–H) LACE assay on the frontal sections of Ndst mutants showed a gradual loss of HS-dependent Fgf-Fgfr association in central retinae isolated from Ndst mutant E10 to E13 mice (arrows). (I–L) LACE signals were also diminished in Pax2-positive Ndst mutant optic stalks on sagittal sections.
Figure 5
Figure 5. MAPK signaling defects in Ndst mutant are partially rescued by Kras
(A–H) In Ndst1/Ndst2 mutants, phospho-ERK was downregulated in central retina, and Pax2 expression was also found to be strongly reduced in the putative optic disc region (E and F, arrows indicate the missing optic discs). As a result, the NF-165-expressing axons projected to the sub-retinal space, failing to reach the optic stalk (F and G), and the optic nerve was missing in adult animal (H). (I–L) Partial rescue of developmental defects by constitutive Kras signaling in Ndst mutant mice. Notice that phospho-ERK was recovered to the wild type level (I, arrow) and Pax2-positive cells were detected in the optic disc and stalk regions (J and K, arrows). Partially restored formation of the optic nerve could therefore be observed in Ndst/Kras compound mutants (L).
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