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First published online 15 November 2006
doi: 10.1242/dev.02679

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Heparan sulfate biosynthetic gene Ndst1 is required for FGF signaling in early lens development

¹ Department of Medical and Molecular Genetics, Indiana University of Medicine, Indianapolis, IN 46202, USA.
² Department of Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
³ Department of General Zoology and Genetics, Westfälische Wilhelms-Universität Münster, Schlossplatz 5, 48149 Münster, Germany.

View larger version (59K): [in this window] [in a new window]   Fig. 1. Ndst1 expression during lens development. (A) Ndst1 is expressed in the developing eye. RNA in situ hybridization was performed on mouse embryonic sections. Lens placodes at E9.5 and lenses at E12.5 were both stained with a Ndst1 antisense probe. As a control, samples were incubated with a Ndst1 sense probe. The Pax6 antisense probe specifically stained the developing lens placode (LP) and optic vesicle (OV) at E9.5, and lens epithelium (LE) and retina (RE) at E12.5. (B) RT-PCR analysis of Ndst gene expression in the lens and retina at E12.5. At this stage, only Ndst1 (N1), Ndst2 (N2) and mitochondria ribosomal subunit L19 were detected in lens mRNA by RT-PCR, whereas all four Ndst genes were expressed in the retina. No signal was detected in the absence of reverse transcriptase (- RT). N3, Ndst3; N4, Ndst4.

View larger version (57K): [in this window] [in a new window]   Fig. 2. Ndst1-mutant ocular phenotypes. (A) The range of ocular phenotypes observed in E12.5 to E17.5 Ndst1KO/KO embryos. (B-I) E13.5 Ndst1 embryos and eye sections showing Pax6 RNA in situ staining. Severe lens-developmental defects were observed in E13.5 Ndst1-mutant embryos (D-I), ranging from small or absent lenses to a complete lack of eyes. (J-S) Retinal patterning in the Ndst1 mutant. RNA in situ hybridization was performed on E14.5 embryos. Sox2, Six3, Hes1, Math5 and BF2 were expressed in both wild-type and Ndst1KO/KO retinae. Notice the lack of lenses in Ndst1KO/KO eyes. KO/KO, homozygous Ndst1-knockout embryos; L, lens.

View larger version (37K): [in this window] [in a new window]   Fig. 3. Lens cell proliferation defects in Ndst1 mutants. (A) Homozygous Ndst1-knockout embryos and wild-type controls were stained for BrdU (red), and the nuclei were counterstained with Hoechst at the 24-, 30- and 34-somite stages. (B) Cell proliferation was quantitated as the ratio of BrdU-positive cells versus Hoechst-positives cells at different stages of development [26- to 29-somite (s) stages; 30- to 33-s stages; and 34- to 38-s stages]. There was a consistent reduction of cell proliferation in Ndst1-mutant lenses compared with wild type (Student's t-test: 26- to 30-somite stages, P<0.001; 30- to 34-somite stages, P<0.01; 34- to 38-somite stages, P<0.001. At least four embryos were analyzed for each genotype at each stage). (C) Lack of apoptosis defects in Ndst1-mutant lens vesicles. TUNEL staining in Ndst1 mutants was normal in the lens vesicle (arrows), but increased in periocular mesenchyme (arrowheads). KO/KO, homozygous Ndst1-knockout embryos; WT, wild type.

View larger version (90K): [in this window] [in a new window]   Fig. 4. Expression of lens-specific genes in Ndst1 mutants. (A-F) At the 26- to 28-somite stage, Pax6 and Six3 were detected in the lens placode (LP), but the level of their expression was reduced in severely affected Ndst1 mutants (arrowhead). (G-O) Molecular defects in Ndst1-mutant lens vesicles at the 35-somite stage. As the lens vesicles invaginated, expression of AP2, Prox1 and A crystallin was downregulated in severely affected mutant lens vesicles (arrowheads in I,L,O). Notice that AP2 expression was still detectable in the overlying ectoderm and Pax6 expression was not perturbed. OV, optic vesicle; LP, lens placode; LV, lens vesicle; RE, retina.

View larger version (87K): [in this window] [in a new window]   Fig. 5. BMP- and Wnt-signaling were unaffected by Ndst1 inactivation. (A) Detection of BMP signaling by Smad1-P immunohistochemistry. Smad1-P was observed in E9.5 wild-type nasal mesenchyme (arrow) and branchial arches (arrowhead), but not in Bmp4-mutant embryos. (B) Smad1-P and Smad2-P expressions were not affected in Ndst1-mutant lenses. In both wild-type and mutant embryos, Smad1-P and Smad2-P was expressed at similar levels in the lens. The same section used for Smad1-P staining was also probed with Pax6 and Pax2 antibodies to visualize the lens vesicle. Arrows indicate lens placode; arrowheads indicate lens vesicle. (C) Lack of genetic interaction between Bmp4 and Ndst1. Addition of the Bmp4LacZ allele did not affect the lens phenotype in Ndst1-mutant eyes (upper panels). Furthermore, Bmp4 expression, as indicated by a knock-in LacZ reporter, was unchanged in the Ndst1 mutant (lower panels). (D) Canonical Wnt signaling indicated by TOPGAL reporter activity was not perturbed by Ndst1 inactivation during lens development. KO/KO, homozygous Ndst1-knockout embryos.

View larger version (59K): [in this window] [in a new window]   Fig. 6. Disruption of heparan sulfate synthesis in Ndst1 mutants. (A) Specificities of Hepss-1, 10E4 and 3G10 heparan sulfate antibodies. (B) Loss of sulfation of heparin sulfate in KO/KO embryos. Hepss-1 and 10E4 antibodies were specific for heparan sulfate. Their staining in the developing lens was lost both in heparitinase I-treated (+ Heparitinase) wild-type embryos and in Ndst1 mutants. By contrast, staining by 3G10 antibody detected the heparan sulfate stub region after heparitinase I cleavage. Staining was observed in both wild-type and mutant embryos. KO/KO, homozygous Ndst1-knockout embryos.

View larger version (38K): [in this window] [in a new window]   Fig. 7. Reduction of FGF and FGFR binding to the Ndst1 lens. (A) Reduced FGF2 binding to Ndst1-mutant embryos. Biotinylated FGF2 (1:10,000 dilution) was incubated with lens sections and assayed by immunohistochemistry in wild-type embryos. Significant FGF2 binding in the Ndst1 mutant was observed only with high concentrations of FGF2 (1:1000 dilution). (B) Diminished FGF-FGFR binding on Ndst1-mutant lenses. Ndst1-mutant lenses exhibited reduced binding to FGF8b-FGFR2c and FGF8b-FGFR3c, whereas FGF19-FGFR4 remained the same for wild-type and mutant embryos. (Arrow indicates weaker staining in retina; arrowhead indicates strongly reduced staining in lens.) (C) Requirement of Ndst1 for multiple FGF-FGFR interactions. Ndst1 mutation disrupts some of the FGF-FGFR interactions on the cell surfaces of the lens (+), but not others (-). *Weak binding. NB, no binding. KO/KO, homozygous Ndst1-knockout embryos.

View larger version (57K): [in this window] [in a new window]   Fig. 8. Loss of FGF signaling in Ndst1-mutant lenses. (A) Specific Erk-P staining in the developing lens. Erk-P was detected in control lenses, but not in embryos treated with MEK- or FGFR-inhibitors. (B) Loss of Erk-P expression in Ndst1-mutant lenses. Erk-P immunostaining was decreased in the Ndst1-mutant lens placode (arrowhead) and lens vesicle (arrow), whereas Pax6 and Pax2 expressions were preserved. (C) The FGF-responsive transcription factor ERM was downregulated in the E10.5 Ndst1 mutant. RNA in situ hybridization showed that ERM was expressed in wild-type, but not in Ndst1-mutant, lenses. The lens vesicle was identified by Pax6 staining the same section. KO/KO, homozygous Ndst1-knockout embryos.