Spatial and temporal expression of heparan sulfate in mouse development regulates FGF and FGF receptor assembly

Abstract

Heparan sulfate (HS) interacts with diverse growth factors, including Wnt, Hh, BMP, VEGF, EGF, and FGF family members, and is a necessary component for their signaling. These proteins regulate multiple cellular processes that are critical during development. However, a major question is whether developmental changes occur in HS that regulate the activity of these factors. Using a ligand and carbohydrate engagement assay, and focusing on FGF1 and FGF8b interactions with FGF receptor (FR)2c and FR3c, this paper reveals global changes in HS expression in mouse embryos during development that regulate FGF and FR complex assembly. Furthermore, distinct HS requirements are identified for both complex formation and signaling for each FGF and FR pair. Overall, these results suggest that changes in HS act as critical temporal regulators of growth factor and morphogen signaling during embryogenesis.

Figure 1.

Binding and activation of FR2c and FR3c by FGF1-heparin and FGF8b-heparin. (A) Percent FRAP binding to HABs under the conditions shown. (B) Numbers of BaF3 cells expressing either FR2c or FR3c after 48 h in culture medium with the indicated treatments.

Figure 2.

FGF8b and FR2cAP assemble with E18 HS, but not E8.5 HS. (A–H) Composite pictures of serial sagittal sections of E18 heart (atrium, At; and ventricle, Ve); lung (Lu), diaphragm (Di), and liver (Li) are shown. (I–P) Sagittal sections of E8.5 mouse embryos are shown as composite images (H, head; He, heart; and T, tail). (A and I) Total HS distribution is identified by mAb 3G10 after treatment with heparin lyases. Sections are incubated with either (B and J) 100 nM FR2cAP or (F and N) 100 nM FR3cAP alone or in the presence of (C, G, K, and O) 30 nM FGF1 or (D, H, L, and P) 30 nM FGF8b. (E and M) FR3cAP is also incubated in the presence of FGF8b on sections pretreated with heparin lyases. FRAP is visualized with antibody to AP. Each treatment was repeated at least three times using different embryos for both stages of development. Bars, 500 μm.

Figure 3.

Global changes in HS-mediated assembly of FGF1-FR3cAP and FGF8b–FR2cAP complexes in E9.5 and E12 mouse embryos. Composite images of sagittal sections of (A–H) E9.5 and (I–P) E12 embryos are shown. Detection of (A and I) total HS with mAb 3G10; (B and J) binding of 100 nM FR2cAP alone; (C and K) binding of FR2cAP in the presence of 30 nM FGF1 or (D and L) 30 nM FGF8b; (F and N) 100 nM FR3cAP alone; (G and O) binding of FR3cAP in the presence of 30 nM FGF1 or (H and P) 30 nM FGF8b. (E and M) Sections pretreated with heparin lyases and incubated with 100 nM FR3cAP and 30 nM FGF8b. He, heart; Li, liver; BA, branchial arch; and Ne, neuroepithelium. Experiments were repeated at least three times with different embryos to confirm the results. Bars, 500 μm.

Figure 4.

Heparin competition of FGF–FRAP complexes from HS in E18 mouse embryos. Detection of FR2cAP (A–D and I–L) or FR3cAP (E–H and M–P) binding to E18 lung preincubated with (I–P) 30 nM FGF1 or (A–H) 30 nM FGF8b. Incubation of (A, E, I, and M) 100 nM FRAP alone, or with (B, F, J, and N) 50 nM heparin, (C, G, K, and O) 50 nM 2-ODS heparin, or (D, H, L, and P) 50 nM 6-ODS heparin. Bar, 100 μm.

Figure 5.

FGF8b and FR3c form an active signaling complex in the absence of 2- O -sulfation. Assembly of FGF–HS–FRAP complexes on (A–D) E10 Hs2st ^+/+ and (E–H) Hs2st ^−/− embryos and wild-type (I–L) CHO-K1 and (M–P) pgsF-17 cells. mAb 3G10 identification of total HS (A, E, I, and M). Binding of (B, F, J, and N) 100 nM FR3cAP after incubation alone, or with (D, H, L, and P) 30 nM FGF8b. Binding of 100 nM FR2cAP after incubation with (C, G, K, and O) 30 nM FGF8b. Bar, 100 μm. (Q) Quantification of BaF3 cells expressing either FR2c or FR3c after 48 h in the treatments shown. (R) Proliferative response of BaF3 cells expressing FR3c to FGF1 or FGF8b and HS isolated from either CHO-K1 or pgsF-17 cells. (Q and R) Error bars represent the SD for quadruplicate samples in a single experiment, and are representative of three independent experiments.

Figure 6.

FGF1–FR2bAP complex assembly in the absence of 2- O -sulfation. Assembly of FGF–HS–FRAP complexes on (A–D) E10 Hs2st ^+/+ and (E–H) Hs2st ^−/− embryos and wild-type (I–M) CHO-K1 and (N–R) pgsF-17 cells. Detection of FRAP after binding of (A, E, J, and O) 100 nM FR2bAP alone or (B, F, K, and P) 100 nM FR2bAP, (C, G, L, and Q) 100 nM FR2cAP, or (D, H, M, and R) 100 nM FR3cAP in the presence of 30 nM FGF1. FGF1 binding alone (I and N) or in the presence of FR2b (K and P, insets) or FR2c (L and Q, insets) is detected. Formation of FGF1 and FR2bAP complexes is detected in (S) E18 mouse lung alone or in the presence of (T) 50 nM heparin, (U) 50 nM 2-ODS heparin, or (V) 50 nM 6-ODS heparin. (W) The proliferative response of BaF3 cells expressing FR2b to FGF1 and heparin, 2-ODS heparin, or 6-ODS heparin is measured. Bar, 100 μm. Error bars represent the SD for quadruplicate samples in a single experiment, and are representative of three independent experiments.

Figure 7.

FGF1 fails to bind HS in the absence of 2- O -sulfation. Sections are from (A–C) E10 Hs2st ^+/+ or (D–F) Hs2st ^−/− embryos or fixed (G–I) CHO-K1 or (J–L) pgsF-17 cells. Binding of (A, D, G, and J) FR2bAP or (B, E, H, and K) FR2cAP to sections or cells preincubated with 30 nM FGF1 is shown. Bound FR3cAP is detected after incubation of 100 nM FR3cAP on sections or cells preincubated with (C, F, I, and L) 30 nM FGF8b. Bar, 100 μm.