Expression of Spicule Matrix Proteins in the Sea Urchin Embryo during Normal and Experimentally Altered Spiculogenesis

Abstract

During its embryonic development, the sea urchin embryo forms an endoskeletal calcitic spicule. This instance of biomineralization is experimentally accessible and also offers the advantage of occurring within a developmental context. Here we investigate the time course of appearance and localization of two proteins among the four dozen that constitute the protein matrix of the skeletal spicule. SM50 and SM30 have been studied in some detail, and polyclonal antisera have been prepared against them (C. E. Killian and F. H. Wilt, 1996, J. Biol. Chem. 271, 9150-9159). Using these antibodies we describe here the localization and time course of accumulation of these two proteins in Strongylocentrotus purpuratus, both in the intact embryo and in micromere cultures. We also investigate the disposition of the matrix proteins, SM50, SM30, and PM27, in the three-dimensional spicule by studying changes in protein localization during experimental manipulation of isolated skeletal spicules. We conclude that SM50, PM27, and SM30 probably play different roles in biomineralization, based on their localization and patterns of expression. It is unlikely that these proteins are solely structural elements within the mineral. SM50 and PM27 may play a role in defining the extracellular space in which spicule deposition occurs, while SM30 may play a role in secretion of spicule components. Finally, we report on the effects of serum on expression of some primary mesenchyme-specific proteins in micromere cultures; withholding serum severely depresses accumulation of SM30 but has only modest effects on the accumulation of other proteins.

The dynamics of secretion during sea urchin embryonic skeleton formation

“…Immunocytochemical staining of SpSM30B and SpSM50 in PMC bodies is diffuse and is not punctate, consistent with their localization in numerous small vesicles. Only very small amounts of SpSM30B, a prominent spicule matrix protein, are located on the surface of the spicule, and static images of immunostaining usually only show protein in the primary mesenchyme cell bodies [15]. The GFP tagged SpSM30B shows a similar behavior, appearing mainly in cell bodies, and in favorable instances showing a clear perinuclear intracellular location on the side of the nucleus facing the spicule, consistent with a predestined vectorial secretion.…”

“…However, in a few instances eGFP-SM30 fluorescence could be seen in filopodial cytoplasm near expressing cells (figure 2E); this example (2E) is a spicule in a larva of Lytechinus pictus, indicating that the eGFP-SM30 fusion transgene is expressed with fidelity in another species, as was the eGFP-SM50 fusion transgene (data not shown). Most SpSM30B is found occluded within the mineral [15] and GFP fluorescence is probably less visible viewed, as it were, through a calcite shroud. We sometimes could see fluorescence of this occluded protein within the exposed mineral when a spicule was inadvertently broken (data not shown).…”

“…Some fluorescence in filopodial cytoplasm is discernable (see figure 2A). SpSM50 is known to surround the spicule, as well as embedded within it [15]. eGFP-SM30 fusion protein expression is also limited to PMCs and displays a mosaic pattern of expression within the PMCs.…”

“…Fluorescence does not spread throughout the PMC syncytial array. Fluorescence of syncytial cytoplasm and spicules is faint, just as is the case when immunostaining procedures are used to localize endogenous SpSM30B protein [15]. However, in a few instances eGFP-SM30 fluorescence could be seen in filopodial cytoplasm near expressing cells (figure 2E); this example (2E) is a spicule in a larva of Lytechinus pictus, indicating that the eGFP-SM30 fusion transgene is expressed with fidelity in another species, as was the eGFP-SM50 fusion transgene (data not shown).…”

“…The SpSM50 protein is an integral matrix protein that has a basic pI and is not glycosylated. Its promoter has been characterized [13] and the protein has been shown to be present and occluded within the calcite as well as present on the surface of the mature spicule [5,14,15]. Deposition of SM50 is essential for spicule elongation [16].…”