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. 2001 Sep 25;98(20):11347-52.
doi: 10.1073/pnas.201418298.

Evidence that the WNT-inducible growth arrest-specific gene 1 encodes an antagonist of sonic hedgehog signaling in the somite

C S Lee  1 , L Buttitta, C M Fan
Affiliations

Evidence that the WNT-inducible growth arrest-specific gene 1 encodes an antagonist of sonic hedgehog signaling in the somite

C S Lee et al. Proc Natl Acad Sci U S A. .

Abstract

The dorsal-ventral polarity of the somite is controlled by antagonistic signals from the dorsal neural tube/surface ectoderm, mediated by WNTs, and from the ventral notochord, mediated by sonic hedgehog (SHH). Each factor can act over a distance greater than a somite diameter in vitro, suggesting they must limit each other's actions within their own patterning domains in vivo. We show here that the growth-arrest specific gene 1 (Gas1), which is expressed in the dorsal somite, is induced by WNTs and encodes a protein that can bind to SHH. Furthermore, ectopic expression of Gas1 in presomitic cells attenuates the response of these cells to SHH in vitro. Taken together, these data suggest that GAS1 functions to reduce the availability of active SHH within the dorsal somite.

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Figures

Figure 1
Figure 1
SHH-N binds to COS cells expressing GAS1. (A) Western blots using Abs to AP (anti-AP), to Fc (anti-Fc), and to SHH-N (anti-SHH-N, 5E1) confirmed that the SHH-N-AP and SHH-N-Fc fusion proteins were of the expected sizes. con (control) and AP, media from 293EBNA cells transfected with vector without insert and vector with AP. As controls, 50 ng of AP and human IgG (Ig) was used. Markers are in kDa. (B) (Left) COS cells transfected with pcDNA3 did not bind AP and bound low levels of SHH-N-AP. COS cells transfected with pcDNA3-Gas1 did not bind AP but did bind SHH-N-AP. (Right) Labeling of SHH-N-Fc and GAS1 on the same cells. (Upper) Cells expressing GAS1 (red, detected by anti-GAS1 and Cy3-2° Abs) do not bind human IgG (Ig, green, detected by FITC-anti-Fc). (Lower) Cells expressing GAS1 bind SHH-N-Fc; 1 μg/ml of fusion ligands was used for binding. (Scale bar = 10 μm.) (C) GAS1-SHH-N-AP binding was competed by the simultaneous addition of a 50-fold excess of recombinant SHH-N or 5E1, but not 9E10 Ab. −, no other reagent was included. (Scale bar, 100 μm.)
Figure 2
Figure 2
SHH-N binds GAS1 and the binding depends on the N-glycosylation of GAS1. (A) GAS1 and SHH-N immunoprecipitate each other. (Left) GAS1 expressed on 293EBNA cells was precipitated by SHH-N-Fc but not by IgG (Ig) with protein A. The supernatant (supe) was lysate containing unbound GAS1. (Right) GAS1-HA precipitated recombinant SHH-N in the presence of anti-HA. The supernatant contained the unbound SHH-N. GAS1-HA and SHH-N (arrowheads) were detected by Western blots. (B) The binding data (Left) and Scatchard plot (Right) show the Kd between SHH-N-AP and GAS1 to be 6.1 nM. (C) (Left) Cells expressing wild-type mouse GAS1 (wt), human GAS1 (hGas1, gray), and chick GAS1 (cGas1, slashed lines) display SHH-N-AP binding. The binding was reduced after 25 ng/ml of tunicamycin treatment for 4 h (+T). Change of the first N-glycosylation site of GAS1 to A, D, or Q (N1A, N1D, and N1Q, respectively) reduced SHH-N-AP binding. (Right) N1A, N1D, N1Q, or tunicamycin-treated proteins were produced at the ≈50% level of the wt GAS1 and were detected on the cell surface by live cell labeling. Tunicamycin-treated GAS1 migrated to the same position as N1A, N1D, and N1Q (black line). GAS1 with the second predicted glycosylation site changed to a proline (N2P) behaved identically to the wild type.
Figure 3
Figure 3
Gas1 expression is associated with the Shh and Ihh expression centers and complementary to Ptc1 expression. Comparative studies were carried out with radioactively labeled or digoxigenin-labeled Shh, Ihh, Ptc1, and Gas1 probes for in situ hybridization on adjacent sections (A–C, G–I, M–R) or whole-mount embryos (D–F, J–L). The embryo stages and tissues are labeled to the left. (A–C) Transverse sections through E9.5 trunk. nc, notochord; nt, neural tube; fp, floorplate; sc, sclerotome; dm, dermomyotome; lp, lateral plate. (D–F) Dorsal views of E10.5 hindlimbs. ZPA, zone of polarizing activity. (G–I) horizontal sections of E10.5 head. tv, telencephalic vesicle; 3v, third ventricle; 4v, fourth ventricle; hb, hindbrain. (J–L) E12.5 vibrissae. wf, whisker follicles. (M–O) E14.5 eyes. gc, ganglionic cells; nr, neural retina; rpe, retinal pigmented epithelium; pom, perioptic mesenchyme; cb, cilliary body. (P–R) E13.5 hand plate. c, condensing chondrocyte; prc, perichondrium; pm, perimesenchyme; tend, future tendon region. Note that P is hybridized with the Indian hedgehog (Ihh) probe.
Figure 4
Figure 4
Gas1 expression in the paraxial mesoderm is regulated by WNT and SHH. (A) E9.5 mouse psm explants were cultured in collagen gels. The inductive tissues were isolated from E2.5 chick. (Left to Right) psm was cultured alone, next to a notochord (nc), a ventral neural tube (vnt), a dorsal neural tube (dnt), or inside a surface ectoderm (se). Dashed lines, the boundary between psm and the chick tissues. Explants were assessed for Gas1 expression. (B) (Left to Right) psms were cocultured with control COS cells (COS-con) or cells expressing SHH-N (COS-SHH-N), BMP4/Furin (COS-BMP4/Furin), and WNT1 (COS-WNT1). Explants were assessed for Gas1 induction. Four samples are represented by each image. (C) Parental 3T3 cells (3T3-con) did not induce Gas1. WNT1-expressing cells (3T3-WNT1) induced Gas1 over 200 μm. The induction was reduced in the presence of 500 ng/ml SHH-N. WNT3A- and WNT4-expressing 3T3 cells also induced Gas1.
Figure 5
Figure 5
GAS1 renders psm resistant to SHH-N. (A) Cytomegalovirus-driving GFP (GFP, as control) and Gas1-internal ribosomal entry site-GFP (GAS1-GFP) were constructed in pAdeno-X vector. (B) BrdUrd incorporation. Explants were cultured in 10 μM BrdUrd for 6 h after infection. Anti-BrdUrd and TRITC-2° Abs were used. GAS1-GFP cells (green) incorporated BrdUrd (red) readily (overlap, yellow). (C) psm cells infected with GAS1-GFP did not respond to SHH-N (50 ng/ml)-induced proliferation but responded to basic fibroblast growth factor (10 ng/ml). (Right) The graph of BrdUrd counts of each treatment. The bars represent standard errors. (D) Pax1 and Ptc1 induction was assessed by quantitative reverse transcription–PCR. psms were infected with virus for 20 h, and SHH-N ranging from 12.5 ng/ml to 400 ng/ml was added. β-actin was used as a control. (Lower Left) GAS1-GFP and GFP infection did not repress Pax1 and Ptc1 in mature somites (somIV–VII). (Lower Right) The amplification ranges for the quantitative reverse transcription–PCR of Ptc1, Pax1, and β-actin. The midamplification cycles were used. PCR products were resolved on 2% agarose gels, stained with ethidium bromide, and quantified by imagequant.
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References

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