Cell-substrate interactions during sea urchin gastrulation: Migrating primary mesenchyme cells interact with and align extracellular matrix fibers that contain ECM3, a molecule with NG2-like and multiple calcium-binding domains

Abstract

The migratory primary mesenchyme cells (PMCs) of the sea urchin embryo are a model experimental system for the analysis of cell-extracellular matrix (ECM) interactions. Although the behavior of PMCs during gastrulation has been analyzed in considerable detail, it has proven difficult to identify specific substrate molecules with which these cells interact. Here, using a new monoclonal antibody (2.5C4) generated by an in vitro immunization procedure, we show that migrating PMCs interact with a distinct class of ECM fiber. The 2.5C4-positive fibers are distributed in a vegetal (high) to animal (low) gradient on the basal surface of the ectoderm. Three observations indicate that PMC filopodia interact directly with the fibers: (1) During gastrulation, 2.5C4-positive fibers gradually become oriented in a prominent circumferential belt that corresponds precisely to the position of the subequatorial PMC ring. (2) This fiber pattern is blocked by microsurgical removal of PMCs but is restored if PMCs are reintroduced into the embryo. (3) Examination of immunostained embryo whole mounts by confocal microscopy reveals a striking association between PMC filopodial roots and foci of fiber bundling. Double-immunostaining experiments using 2.5C4 and antibodies against previously identified matrix constituents show that the protein ECM3 is a component of the fibers. We have determined the complete amino acid sequence of ECM3 and find that this large protein (3103 amino acids) consists of an N-terminal domain similar to the mammalian chondroitin sulfate proteoglycan core protein NG2, a central region composed of five tandem repeats of a domain contained within the regulatory Ca2+-binding loop of Na+-Ca2+ exchange proteins, and a C-terminal region with no homology to known proteins. The general structure of ECM3 is similar in several respects to that of a sponge protein, MAFp4. MAFp4 is a major component of aggregation factor, an ECM complex that mediates the calcium-dependent, species-specific sorting of sponge cells. These studies establish ECM3 as a strong candidate for a PMC substrate molecule and point to several possible mechanisms by which interactions between PMC filopodia and ECM3-containing fibers could provide guidance information to migrating PMCs.

Growth factor-mediated mesodermal cell guidance and skeletogenesis during sea urchin gastrulation

“…Modifications were introduced to label PMCs with the 6e10 mAb (Hodor et al, 2000), prior to nuclear labeling with 0.2 mg/ml Hoechst stain. Embryos were observed by confocal microscopy as described above.…”

Genome-wide analysis of the skeletogenic gene regulatory network of sea urchins

“…During their migration, PMCs interact with extracellular matrix (ECM) fibers that contains the sea urchin ortholog of vertebrate Frem2 (Hodor et al, 2000). Frem2 and related proteins have been implicated in epithelium-mesenchyme adhesion and mutations in these genes underlie Fraser's syndrome (Smyth and Scambler, 2005).…”

Tissue morphogenesis and vascular stability require the Frem2 protein, product of the mouse myelencephalic blebs gene

“…Thus, Frem2 protein is positioned so as to allow it to mediate tissue cohesion during specific rearrangements. Its closest known homologue, the sea urchin protein ECM3, appears to play precisely this role, because its localization within the ECM is reorganized by migrating cells, which then continue to associate with the resulting ECM3-containing fibers (10).…”

“…The only other data relevant to the Fras͞Frem family comes from studies of the sea urchin Frem2 orthologue, ECM3. During gastrulation, the ECM3 protein is localized in a fibrous mesh; primary mesenchyme cells appear to associate with and reorder this structure during their dorsal migration (10). Thus, ECM3 appears to act as a substrate for cell migration.…”