PDGF-A interactions with fibronectin reveal a critical role for heparan sulfate in directed cell migration during Xenopus gastrulation

Abstract

Platelet-derived growth factor (PDGF) signaling is essential for processes involving cell motility and differentiation during embryonic development in a wide variety of organisms including the mouse, frog, zebrafish, and sea urchin. In early Xenopus laevis embryos, PDGF-AA provides guidance cues for the migration of anterior mesendoderm cells as they move across a fibronectin-rich extracellular matrix. The long form of PDGF-A includes a positively charged carboxyl-terminal retention motif that can interact with the extracellular matrix and heparan sulfate proteoglycans (HSPGs). In this study we demonstrate that PDGF-AA binds directly to fibronectin and that this association is greatly enhanced by heparin. The PDGF-AA-fibronectin binding occurs across a broad range of pHs (5.5-9), which is significant because the PDGF-guided migration of Xenopus mesendoderm cells occurs under basic extracellular conditions (pH 8.4). We further demonstrate that endogenous HSPG's are required for the PDGF-AA-guided mesendoderm movement, suggesting an in vivo role for HSPGs in mediating the interaction between PDGF-AA and fibronectin.

Fig. 1.

PDGF-AA binding to fibronectin is increased by pretreatment of the fibronectin with heparin. (A) Binding of ¹²⁵I-PDGF-AA, in the presence or absence of excess (1 μg/mL) PDGF-AA, to 40 nM fibronectin was tested at pH 7.5, without (FN) or with (FN/Hep) pretreatment of the fibronectin with 1 μg/mL heparin. Control (Con) wells included ¹²⁵I-PDGF-AA in the absence of excess growth factor. (B) Adsorbed fibronectin (40 nM) without (solid line) or with 1 μg/mL heparin pretreatment (dashed line), was incubated with 0.35 nM to 34.5 nM ¹²⁵I-PDGF-AA and the amount bound determined (y axis). Note PDGF-AA effectively self competes and its binding to fibronectin alone reaches a maximum of 0.42 nM, but heparin pretreatment increased this to 1.07 nM. (C) PDGF-AA binding to fibronectin at pH 5.5–7.5 was examined without or with pretreatment of the fibronectin with 100 μg/mL heparin. Wells contained no fibronectin (-FN; white bars), fibronectin (FN; gray bars), or pretreated fibronectin (FN/Hep; black bars). (D) Binding of ¹²⁵I-PDGF-AA to fibronectin was examined over an increasing concentration of heparin 0.1–100 μg/mL at pH 7 (white bars), 8 (gray bars), or 9 (black bars). (E) PDGF-AA binding to three fibronectin fragments, 40 kDa, 70 kDa, and 120 kDa, was examined without (−) or with (+) 1 μg/mL heparin pretreatment. Full-length fibronectin (FN-FL) and all fibronectin fragments were used at equimolar concentrations (40 nM). No fibronectin (−FN). Data are representative of at least two independent experiments.

Fig. 2.

Heparinase III treatment disrupts the directed migration of embryonic mesendoderm cells. (A and B) Ex vivo assay for directed cell movement of head mesendoderm cells. (A) Preparation of conditioned substrata. Schematic of a sagital section of a stage 10 Xenopus laevis embryo. The cell sheet (blastocoel roof) that supports the migration of mesendoderm cells in vivo is removed (short arrows indicate dissection) and placed matrix-side down on a tissue culture dish. The position and orientation of the tissue is marked on the dish. After 2 h, the tissue is removed leaving the deposited ECM. (B) Directed Migration Assay. An explant of anterior mesendoderm (circle) is dissected at stage 10.5 and placed on the ECM at the mid point between the blastocoel lip (BL) and animal pole (AP) marks. The explant position is recorded immediately and 1 h later. (C) Following the migration assay, the conditioned substrata are subjected to immunocytochemistry for fibronectin. (D) RT-PCR analysis of anterior mesendoderm explants. (E–G) The position of each mesendoderm explant after 1 h is plotted with all starting positions superimposed on the origin. Positive y values indicate movement toward the animal pole, the normal direction of migration. (E) Control, (F) 0.1 U/mL heparinase III included throughout the experiment. (G) ECM treated with 0.1 U/mL heparinase III for 45 min immediately following its deposition. (H) Bar graph of data in E–G. Direction of movement: animal pole (black bars), blastopore lip (gray bars), lateral (moved <15 μm on the y axis but >15 μm on the x axis; hatched bars), stalled (moved less that 15 μm on both axes; white bars). (I) Graph indicating the absence (black bars) or presence (hatched bars) of apoptotic cells in the blastocoel cavity following microinjection of vehicle, heparinase III, or heat-inactivated heparinase III. Dead embryos (white bars). All experiments were repeated at least three times. [Scale bar, 10 μm (E; applies to frames C–E.)]