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First published online 12 December 2007
doi: 10.1242/dev.014829

Development 135, 301-310 (2008)
Published by The Company of Biologists 2008
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Bud specific N-sulfation of heparan sulfate regulates Shp2-dependent FGF signaling during lacrimal gland induction

Yi Pan1, Christian Carbe1, Andrea Powers1, Eric E. Zhang2, Jeffrey D. Esko3, Kay Grobe4, Gen-Sheng Feng2 and Xin Zhang1,*

1 Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
2 Programs in Signal Transduction and Stem Cells and Regeneration, Burnham Institute for Medical Research, La Jolla, CA 92037, USA.
3 Department of Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
4 Department of General Zoology and Genetics, Westfälische Wilhelms-Universität Münster, Schlossplatz 5, 48149 Münster, Germany.


Figure 1
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  		Fig. 1. Le-Cre is specifically expressed in lacrimal gland. (A-C) The Le-Cre transgene GFP expression. GFP expression from the Le-Cre transgene was first visible in the E14.5 lacrimal gland bud (arrowhead), and it continued to be present in the elongated lacrimal gland (arrowhead) at E16.5 and lacrimal gland lobes (arrow) at P0. (D-F) Co-expression of Pax6, GFP and Cre in the Le-Cre embryo. In contrast to the nuclear Pax6 expression in retina and lacrimal gland (D), cytoplasmic GFP and nuclear Cre were found exclusively in the lacrimal gland bud (E,F). (G-I) The Le-Cre transgene Cre activity. Cre recombinase activity in the Le-Cre transgene was determined by crossing with R26R mice, which marked all the Cre-expressing cells and their progenies with lacZ expression identifiable by X-gal staining (G). In P0 lacrimal gland sections, all X-gal-stained cells resided in the Pax6 positive epithelium, demonstrating the restricted expression of the Le-Cre transgene.

 

Figure 2
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  		Fig. 2. Heparan sulfate modification is required for lacrimal gland development. (A-C) Total heparan sulfate identified by the 3G10 antibody (red) was ubiquitously expressed in lacrimal gland (arrow) and the surrounding mesenchyme. HS, heparan sulfate. (D-F) Sulfated heparan sulfate recognized by the 10E4 antibody (red) exhibited restricted expression, with the strongest staining at the tip of Pax6-positive lacrimal gland bud (arrow). (G-I) Ndst1 is expressed in the lacrimal gland bud as marked by Pax6 staining (arrowhead). (J-L) Ndst1-/- animals failed to develop lacrimal gland as shown by aceto-carmine staining and GFP expression (arrow). p, posterior stalk; t, tip.

 

Figure 3
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  		Fig. 3. Epithelial and mesenchymal specific knockout of Ndst1. (A-D) Tissue specific deletion of Ndst1 in the lacrimal gland bud was demonstrated by 10E4 staining. 10E4 expression is present in the tip of the lacrimal gland bud in wild-type and Ndst1flox/flox;Wnt1-Cre mutant (A, B, arrows), but not in Ndst1flox/flox;Le-Cre mutant (C, arrow). All lacrimal gland 10E4 staining was lost in the Ndst1flox/flox; Wnt1-Cre;Le-Cre mutants (D). (E-G) Visualized by aceto-carmine staining, only the wild type and Ndst1flox/flox;Wnt1-Cre mutant exhibited lacrimal glands at birth; the Ndst1flox/flox;Le-Cre mutant did not (arrowheads). (H-K) Lacrimal gland defects in Ndst1 and Ndst1/2 double mutants. The lacrimal gland was either severely stunted or completely missing in the Ndst1flox/flox;Le-Cre mutants, as shown by GFP expression (I,J; arrow). In Ndst1/2 double mutants (Ndst1flox/flox;Ndst2-/-;Le-Cre), no lacrimal gland was ever observed (K). (L) The percentage of lacrimal gland phenotypes in Ndst mutants. The total numbers of eyes examined are shown in the last column.

 

Figure 4
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  		Fig. 4. Lacrimal gland budding defects in Ndst1 mutants. (A,B) Lack of lacrimal gland bud in the Ndst1flox/flox;Le-Cre mutant at E14.5 (arrowhead). (C-F) The periocular expressions of Fgf10 (C,D, arrows) and Fgfr2b (E,F, arrows) were unchanged in mutant embryos when compared with wild type. (G,H) In the Ndst1 mutants, total heparan sulfate stained by the 3G10 antibody was also unchanged (arrow). (I,J) The Ndst1 mutant lost 10E4 staining for sulfated heparan sulfate in the fornix but not in the far side of conjunctival epithelium (I,J, arrows and arrowheads). The conjunctival epithelium is outlined. (K,L) Lack of lacrimal gland budding and BrdU+ proliferating cells in the Ndst1flox/flox;Le-Cre mutants. The lacrimal gland bud in the wild-type and the conjunctival epithelium in mutant embryo are marked by broken lines.

 

Figure 5
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  		Fig. 5. Ndst1 mutation disrupted Fgf10/Fgfr2b interaction and ectopic lacrimal gland budding. (A-F) LACE assay of FGF/FGFR binding at the lacrimal gland buds. Heparan sulfate mediated Fgf10/Fgfr2b and Fgf7/Fgfr2b binding were observed in the tip of wild-type lacrimal gland buds, but not in that of the Ndst1flox/flox;Le-Cre mutant embryos (arrow). By contrast, ubiquitous Fgf1/Fgfr2b was present in both wild-type and mutant embryos (arrowheads). (G-L) Ectopic lacrimal gland budding induced by Fgf10. The control explant developed endogenous lacrimal gland in the presence of BSA-containing beads (G, asterisks). By contrast, Fgf10 beads induced additional ectopic lacrimal gland buds in both control and Ndst1flox/flox;Le-Cre explants (J,K, arrowheads), whereas no buds appeared in the Ndst1/2 double mutants (I,L). (M) Lacrimal budding rate in explant cultures [Fisher's exact test: endogenous budding in control and Ndst1flox/flox;Le-Cre mutants, P<0.001; ectopic budding in control and Ndst1/2 double mutants (Ndst1flox/flox;Ndst2-/-;Le-Cre), P<0.02].

 

Figure 6
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  		Fig. 6. Fgfr2 signaling is required for lacrimal gland development. (A-D) Lacrimal gland development was abrogated in Fgfr2flox/flox;Le-Cre mutants. The control, but not Fgfr2 mutants, showed a mature lacrimal gland at birth (A,B) and a lacrimal bud at E14.5 (C,D, arrow). (E,F) The Fgfr2 mesenchymal knockout did not affect lacrimal gland budding, as shown by aceto-carmine staining (arrowhead). (G,H) Lack of lacrimal gland budding and cell proliferation (arrow) in the Fgfr2flox/flox;Le-Cre mutant conjunctival epithelium (outlined). (I,J) Fgfr2flox/flox;Le-Cre mutants lost heparan sulfate 10E4 staining in the fornix of the conjunctival epithelium (arrow), but not in the ectoderm further away from the eye (arrowhead). The conjunctival epithelia are outlined. (K,L) Phospho-Shp2 expression was present in the control lacrimal gland bud (K, arrow), but lost in the Fgfr2flox/flox;Le-Cre mutants (L, arrowhead).

 

Figure 7
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  		Fig. 7. The Shp2 knockout disrupted ERK signaling in lacrimal gland development. (A,B) Loss of Shp2 staining in the conjunctival epithelium of Shp2flox/flox;Le-Cre mutants (broken lines). (C,D) Loss of cell proliferation and lacrimal gland bud in the Shp2 deletion mutant. (E-H) Shp2 knockout disrupted lacrimal gland development at birth and at E14.5 (arrows). (I,J) Deletion of Shp2 resulted in the downregulation of heparan sulfate 10E4 staining in the fornix of the conjunctival epithelium (arrows). (K-R) Phospho-ERK expressions were present in control lacrimal gland buds (arrows), but not in Shp2, Fgfr2 and Ndst1 mutants at the either E12 or E14 (arrowheads). (S-V) ERM expression was lost in the E14.5 Shp2, Fgfr2 and Ndst1 mutants (arrowheads).

 

Figure 8
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  		Fig. 8. Model of Ndst-Fgfr2-Shp2 signaling in lacrimal gland development. The N-sulfated heparan sulfate proteoglycan (HSPG) catalyzed by Ndst selectively potentiates Fgf10 and Fgfr2 interaction at the lacrimal gland bud. This leads to phosphorylation and activation of the downstream Shp2 protein, which promotes phospho-ERK expression, Pea3/Erm transcription and cell proliferation. In a feedback mechanism, Fgfr2-Shp2 signaling further regulates heparan sulfate modification, thus restricting FGF signaling and cell proliferation within the tip of lacrimal gland bud.

 

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