Hideki Katow

Inhibition of cell migration in sea urchin embryos by β-d-xyloside

Abstract This investigation examines the effect of exogenous xylosides on primary mesenchyme cell behavior in Strongylocentrotus purpuratus embryos. In confirmation of studies in some other species the addition of 2 mM, p-nitrophenyl-β- d -xylopyranoside blocks the migration but not the initial ingression of primary mesenchyme cells. The blastocoel matrix of treated embryos appears deficient in a 15- to 30-nm-diameter granular component that is observed extensively on the basal lamina and on filopodia of migrating primary mesenchyme cells in untreated embryos. Other blastocoel components appear unaffected by ultrastructural criteria. The incorporation of 35SO2−4 per embryo into ethanol precipitates of isolated blastocoel matrices was reduced significantly after xyloside treatment but the distribution of 35SO2−4 after polyacrylamide gel electrophoresis or the glycosaminoglycan composition was unaffected. Chromatography on Sepharose CL-2B demonstrates a reduction in size of sulfated components of the blastocoel. While over 60% of the 35S-labeled material from the blastocoel of normal mesenchyme blastulae is voided from a Sepharose CL-2B column run in a dissociative solvent, only 10% from xyloside treated embryos is voided. Instead, there is a large included peak with Kav of 0.33. This material is acid soluble but cetylpyridinium chloride precipitable. It apparently consists largely of free glycosaminoglycan chains. Based on analysis of chondroitinase ABC digestion products this material consists of 41% chondroitin-6-sulfate and 58% dermatan sulfate. These results are consistent with a role in cell migration for intact chondroitin sulfate/dermatan sulfate proteoglycans in the sea urchin blastocoel matrix.

A fibronectin-related synthetic peptide, ProAlaSerSer, inhibits fibronectin binding to the cell surface, fibronectin-promoted cell migration in vitro, and cell migration in vivo☆

The biological activity of the amino acid sequence consisting of the immediate carboxyl terminus side of the Arg-Gly-Asp-Ser (RGDS) amino acid sequence in the cell-binding domain of intact fibronectin (FN) molecules was examined using synthetic peptides [RGDS, Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro (RGDSPASSKP), Pro-Ala (PA), Pro-Ala-Ser (PAS), Pro-Ala-Ser-Ser (PASS), and Pro-Ala-Ser-Ser-Lys (PASSK)]. These peptides were applied to the primary mesenchyme cells (PMCs) of the sea urchin, Clypeaster japonicus. In vitro immunohistochemistry indicated that the binding of exogenous FN to the PMC surface was inhibited by the peptides RGDSPASSKP and PASS, but not by RGDS, GRGDSP, PA, or PAS. PASS and RGDS introduced into the blastocoel also inhibited PMC migration in vivo. FN-promoted PMC migration in vitro was also inhibited by PASS and RGDS. The present results indicate that the PASS peptide inhibits FN binding to the PMC surface and promotes PMC migration, suggesting that the FN molecule uses the PASS amino acid sequence to bind to the PMC surface and to promote PMC migration in the blastocoel.

Behavior of sea urchin primary mesenchyme cells in artificial extracellular matrices

The primary mesenchyme cells (PMCs) were separated from the mesenchyme blastulae of Pseudocentrotus depressus using differential adhesiveness of these cells to plastic Petri dishes. These cells were incubated in various artificial extracellular matrices (ECMs) including horse serum plasma fibronectin, mouse EHS sarcoma laminin, mouse EHS sarcoma type IV collagen, and porcine skin dermatan sulfate. The cell behavior was monitored by a time-lapse videomicrograph and analysed with a microcomputer. The ultrastructure of the artificial ECM was examined by transmission electron microscopy (TEM), while the ultrastructure of the PMCs was examined by scanning electron microscopy (SEM). The PMCs did not migrate in type IV collagen gel, laminin or dermatan sulfate matrix either with or without collagen gel, whereas PMCs in the matrix which was composed of fibronectin and collagen gel migrated considerably. However, the most active and extensive PMC migration was seen in the matrix which contained dermatan sulfate in addition to fibronectin and collagen gel. This PMC migration involved an increase not only of migration speed but also of proportion of migration-promoted cells. These results support the hypothesis that the mechanism of PMC migration involves fibronectin, collagen and sulfated proteoglycans which contain dermatan sulfate.

Micromere differentiation in the sea urchin embryo : immunochemical characterization of primary mesenchyme cell-specific antigen and its biological roles

Primary mesenchyme cell (PMC)‐specific antigens in developing sea urchin embryos of five different species have been studied by using two different monoclonal antibodies, P4 and B2C2. Like B2C2 in Strongylocentrotus purpuratus (Anstrom et al., 1987) P4 reacted with the N‐linked carbohydrate in Strongylocentrotus intermedius embryo. Although both antibodies recognize the same group of glycoproteins in S. intermedius, P4 epitopes appeared earlier than B2C2 epitopes in Clypeaster japonicus embryo. PMCs of Anthocidaris crassispina blastulae raised in sulfate‐deficient sea water were immuno‐reactive with P4 but not with B2C2, although the embryos raised in normal sea water reacted with both antibodies at similar intensity. These results suggest that the epitopes of P4 and B2C2 are formed by glycosylation and sulfation, respectively. PMCs may display differential modification in their surface glycoprotein synthesis during differentiation. Furthermore, P4 inhibited cultured micromere descendant cells of Hemicentrotus pulcherrimus from attaching to the plastic dishes and forming spicules in vitro without detectable cytotoxic effect. P4‐reactive glycoproteins may play important roles in cell‐substrate interaction and spicule formation.

A New Technique for Introducing Anti‐Fibronectin Antibodies and Fibronectin‐Related Synthetic Peptides into the Blastulae of the Sea Urchin, Clypeaster japonicus.

Migration of primary mesenchyme cells (PMCs) of the sea urchin, Clypeaster japonicus, was examined in vivo by introducing anti‐fibronectin (FN) IgG, and FN‐related synthetic peptides, Gly‐Arg‐Gly‐Asp‐Ser‐Pro‐Cys, Gly‐Arg‐Gly‐Asp‐Ser, and Arg‐Gly‐Asp‐Ser (RGDS) into the blastocoel by a new technique. In this technique the embryos are treated with cytochalasin B (CB) and part of the presumptive PMCs (PPMCs) is removed, leaving a hole in the vegetal plate. Then macromolecules are introduced into the blastocoel through this hole. Their introduction was confirmed by introducing them with polystyrene beads as a marker. The hole closes soon after introduction of the materials, and so these materials remain in the blastocoel. After this treatment, blastulae had fewer PMCs, because of partial loss of PPMCs, but their morphogenesis proceeded normally. Introduction of anti‐FN IgG or the synthetic peptides inhibited PMC migration in vivo, and this inhibition was associated with failure of the PMCs to form cell processes. These results indicate that sea urchin PMCs use the RGDS amino acid sequence in the FN molecule for migration in vivo.

Inhibition of Cell Surface Binding of Fibronectin and Fibronectin‐Promoted Cell Migration by Synthetic Peptides in Sea Urchin Primary Mesenchyme Cells In Vitro

The role of a site of fibronectin molecule in the cell binding and cell migration was examined in vitro using sea urchin primary mesenchyme cells and synthetic peptides that contain a particular amino acid sequence, Arg‐Gly‐Asp‐Ser.

Behavior of Primary Mesenchyme Cells In situ Associated with Ultrastructural Alteration of the Blastocoelic Material in the Sea Urchin, Anthocidaris crassispina

In the blastula of the sea urchin, Anthocidaris crassispina, a small number of primary mesenchyme cells (PMCs) ingressed from the blastocoel wall taking a bottle shape. The majority of the PMCs followed the first group of PMCs. These ingressed without taking the bottle shape, and became round within the blastocoel wall. After ingression, the PMCs migrated as single cells retaining their round cell contour. The average velocity of their migration was 13.3 μm/hr.

Characterization of Vegetal Plate Cells Separated from Cytochalasin B-Treated Blastulae of the Sea Urchin, Clypeaster japonicus

Vegetal plate forming cells (VPCs) of the vegetal plate blastulae of the sea urchin, Clypeaster japonicus, had a layer of microfilaments on the basal side. The VPCs specifically protruded from the embryos after a treatment with 1 μg/ml of cytochalasin B (CB). Based on scanning electron microscopy, unlike other epithelial cells the protruded VPCs possessed neither cilium nor microvilli on their surface. The protruded VPCs were easily separated from the embryos by stirring the embryo suspension with pipette. An in vitro immunohistochemistry using a primary mesenchyme cell (PMC) surface‐specific monoclonal antibody (MAb) raised against PMCs of Strongylocentrotus purpuratus showed that the MAb also specifically bound to the PMCs in mesenchyme blastulae of C. japonicus. The MAb bound in 81% of the separated VPCs in C. japonicus vegetal plate blastulae examined. However, the MAb binding occurred only after the separated VPCs were incubated in artificial sea water (ASW) for at least 1 hr. In the VPC‐deprived embryos, gastrulation occurred after they were transferred to normal ASW. However, the PMCs and the spicules were not formed in these embryos.

Histological distribution of FR-1, a cyclic RGDS-peptide, binding sites during early embryogenesis, and isolation and initial characterization of FR-1 receptor in the sand dollar embryo

A fibronectin‐related synthetic cyclic H‐Cys‐Arg‐Gly‐Asp‐Ser‐Pro‐Ala‐Ser‐Ser‐Cys‐OH (RGDSPASS) peptide (FR‐1) binding site in the embryo of the sand dollar Clypeaster japonicus was specified using dansyl‐labeled FR‐1 (Dns‐FR‐1) and horseradish peroxidase‐labeled FR‐1, and an FR‐1 receptor was isolated using FR‐1‐affinity column chromatography. The FR‐1 introduced to the blastocoel of blastulae inhibited primary mesenchyme cell (PMC) migration in mesenchyme blastulae, and complete gastrulation and spicule differentiation in gastrulae. The Dns‐FR‐1 bound to the entire basal side of the ectoderm in mesenchyme blastulae, and then restricted to the basal side of the ectoderm at the apical tuft region and the vegetal hemisphere in early gastrulae. The cytoplasm of the archenteron also bound to Dns‐FR‐1. In PMC, Dns‐FR‐1 bound to the nucleus and cytoplasmic reticular features. In unfertilized eggs, Dns‐FR‐1 bound to the entire cytoplasm, particularly to the oval‐shaped granules and the nuclear envelope, but only to the cytoplasm after fertilization. Relative molecular mass (Mr) of the FR‐1 ‐binding protein was 240 kDa under non‐reducing conditions and 57 kDa under reducing conditions. The FR‐1 receptor protein bound anti‐sea urchin integrin (Spl) βL subunit antibodies raised against the embryos of Strongylocentrotus purpuratus. Immunohistochemistry showed that the antibody binding site was similar to the histochemical distribution of Dns‐FR‐1. However, Mr of the FR‐1 receptor is distinctively larger than that of the Spl βL subunit.

Behavior and Ultrastructure of Primary Mesenchyme Cells at Sessile Site during Termination of Cell Migration in Early Gastrulae

The behavior and ultrastructure of primary mesenchyme cells at two ventrolateral sessile sites in early gastrulae were examined by time‐lapse videomicroscopy, scanning electron microscopy, and immunotrans‐mission electron microscopy using the sea urchin, Hemicentrotus pulcherrimus and the sand dollar. Clypeaster japonicus. At sessile sites in early gastrulae, PMCs terminated their migration after “touch‐and‐go” behavior, and even after the termination they retained a pulsatile movement. These behaviors indicate that the termination of PMC migration is not due to deprivation of cell motility nor the establishment of firm adhesion between PMCs and the site. PMCs used short cell processes during migration, and extended longer ones during the early period of migration termination. During the final period of migration at the sessile sites, PMCs extended characteristically thin and long cell processes to the basal lamina. These cell processes, as far as present results indicate, never attach to the blastocoel wall cells through the basal lamina. Thus it is indicated that the primary interaction site for PMCs to terminate their migration is the basal lamina.