Gene expression and stress response in sea urchin embryos with skeleton defects caused by magnesium deprivation.

Abstract

Echinoderms have an extensive endoskeleton composed of magnesian calcite, a form of calcium carbonate that contains small amounts of magnesium carbonate and occluded matrix proteins [1]. In the frame of the Biomintec European Project focused on the understanding of basic biomineralization mechanisms for the design of novel strategies in nano-biotechnology, we studied the effects of magnesium deprivation on Arbacia lixula sea urchin embryo development. Embryos were morphologically monitored, evaluating developmental abnormalities at different endpoints (3, 6, 24, 48 and 72 hours). In parallel, the spatial transcriptional levels of a skeleton matrix protein (msp130) and the protein synthesis levels of a signaling protein (p38MAPk) were detected by in situ hybridization (ISH) and Western Blotting (WB). The morphological analysis evidenced a general delay in development soon after the morula stage (6 hours), the absence of biomineral deposition after 24 hours and severe skeleton malformations after 48-72 hours. We observed the ectopic localization of primary mesenchyme cells (PMCs), the only cells in the embryo directing skeleton formation, by immuno-fluorescence with a FITC-conjugated lectin (WGA) able to bind specifically the PMCs. By ISH we detected the msp130 mRNA correctly in PMCs, although it was found persistently expressed even at 48h after fertilization, unlike control embryos where the messenger is down-regulated at this stage. At 48 hours we found high levels of p38MAPk by WB, if compared to control embryos; preliminary experiments showed hsp70 levels similar to controls. Further experiments will be performed to characterize the role of magnesium in the biomineralization process: we have now a toolkit of probes for various skeletogenic-specific genes (msp130, SM30, SM50, p16, p19) [2, 3] that will be used to study their embryonic temporal and spatial expression profiles. The internal calcium content and eventually the calcium carbonate polymorph forms [4] occurring in embryonic spicules in Mg-deprived embryos will be studied in comparison to control embryo. References [1] Matranga V, Bonaventura R, Costa C, Karakostis K, Pinsino A, Russo R, Zito F. Echinoderms as blueprints for biocalcification: regulation of skeletogenic genes and matrices. Prog Mol Subcell Biol (2011) 52:225-48. [2] Pinsino A, Roccheri MC, Costa C, Matranga V. Manganese interferes with calcium, perturbs ERK signaling, and produces embryos with no skeleton. Toxicol Sci (2011) 123:217-230. [3] Costa C, Karakostis K, Zito F, Matranga V. Phylogenetic analysis and expression patterns of p16 and p19 in Paracentrotus lividus embryos. Dev Genes Evol (2012) 222:245-51. [4] Raz S, Hamilton PC, Wilt FH, Weiner S, Addadi L. The transient phase of amorphous calcium carbonate in sea urchin larval spicules: the involvement of proteins and magnesium ions in its formation and stabilization. Adv Mater (2003) 13:480-486.