Characterization and Endocytic Internalization of Epith-2 Cell Surface Glycoprotein during the Epithelial-to-Mesenchymal Transition in Sea Urchin Embryos

Norio Wakayama,Tomoko Katow,Hideki Katow

doi:10.3389/fendo.2013.00112

Abstract

The epithelial cells of the sea urchin Hemicentrotus pulcherrimus embryo express an Epith-2, uncharacterized glycoprotein, on the lateral surface. Here, we describe internalization of Epith-2 during mesenchyme formation through the epithelial-to-mesenchymal transition (EMT). Epith-2 was first expressed on the entire egg surface soon after fertilization and on the blastomeres until the 4-cell stage, but was localized to the lateral surface of epithelial cells at and after the 16-cell stage throughout the later developmental period. However, primary mesenchyme cells (PMC) and secondary mesenchyme cells (SMC) that ingress by EMT lost Epith-2 from their cell surface by endocytosis during dissociation from the epithelium, which was associated with the appearance of cytoplasmic Epith-2 dots. The cytoplasmic Epith-2 retained a similar relative molecular mass to that of the cell surface immediately after ingression through the early period of the spreading to single cells. Then, Epith-2 was completely lost from the cytoplasm. Tyrosine residues of Epith-2 were phosphorylated. The endocytic retraction of Epith-2 was inhibited by herbimycin A (HA), a protein tyrosine kinase (PTK) inhibitor, and suramin, a growth factor receptor (GFR) inhibitor, suggesting the involvement of the GFR/PTK (GP) signaling pathway. These two GP inhibitors also inhibited PMC and SMC spreading to individual cells after ingression, but the dissociation of PMC and SMC from the epithelium was not inhibited. In suramin-treated embryos, dissociated mesenchyme cells migrated partially by retaining their epithelial morphology. In HA-treated embryos, no mesenchyme cells migrated. Thus, the EMT occurs in relation to internalization of Epith-2 from presumptive PMC and SMC.

Highlights

The epithelial-to-mesenchymal transition (EMT) occurs in various processes found in metazoans, such as normal morphogenesis, the mesoderm formation in rabbit embryos [1], and neural crest cell formation in vertebrates [rev
The embryos were fixed in 4%
The anti-Epith-2 monoclonal antibody (mAb) IgG was added to the cell suspension in 24-well plates at 10 or 50 μg/ml and was placed in an incubator at 15°C for 5 h

Summary

Introduction

The epithelial-to-mesenchymal transition (EMT) occurs in various processes found in metazoans, such as normal morphogenesis, the mesoderm formation in rabbit embryos [1], and neural crest cell formation in vertebrates [rev. The EMT has been reported in relation to mesenchyme formation, in sea urchin embryos in which the ingression of primary mesenchyme cells (PMC) occurs at the vegetal plate region of late blastulae [7,8,9,10]. The EMT produces secondary mesenchyme cells (SMC) at the tip of the archenteron in late gastrulae [8, 10, 11]. The EMT is associated with an alteration of cell surface properties such as the retraction of epithelial cell surface-specific Epith-1 protein that is recognized by a monoclonal antibody (mAb), anti-Epith-1mAb, in the sea urchin

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Results

Conclusion