Skeleton growth inhibition in sea urchin embryos Paracentrotus lividus after manganese exposure.

In the sea urchin embryos, skeleton is specified by interactions between PMCs and patterning cues derived from the ectoderm. PMCs use spatial and temporal information to organize the proper animal-vegetal and oral-aboral position and orientation of the two tri-radiate skeletal spicules. Many experiments have demonstrated that exposure to metals, such as lithium, zinc and nickel, can disrupt skeleton patterning information. Here, we have investigated the effects of manganese (Mn) on Paracentrotus lividus embryo development. We found that Mn exposure prevents skeleton growth producing spicule-lacking embryos. Normal skeleton growth was partially rescued after Mn removal from the culture. To determine the effects of Mn exposure on the differentiation of the three germ layers, Mn-exposed embryos were immunostained with 5C7, UH2-95 and 1D5 monoclonal antibodies (Mab) recognizing antigens present on the midgut/hindgut, ciliary band and PMCs, respectively. Ectoderm, endoderm and mesoderm markers were detected in Mn-exposed skeleton-lacking embryos at the appropriate time and in the correct position. By in situ hybridization we analyzed the expression of three genes expressed during PMCs differentiation in Mn-exposed embryos, SM50, SM30 and msp130, encoding two spicule matrix proteins and the cell surface protein detected by 1D5 Mab, respectively. Results showed that: i) SM50 expression was largely normal; ii) SM30 expression was severely reduced and iii) msp130 expression was not down-regulated during development. Activation of p42-44 and p38 MapKs was analyzed by Western blotting in Mn-exposed embryos during their development. We found a persistent phosphorylated state of both MapKs, as proteins levels were only partially modulated during development of Mn-exposed embryos. Results from fluorescent labeling of intracellular calcium, obtained by calcein labelling, and the amount of calcium concentration, determined by atomic absorption spectrophotometer in the same specimens, showed a reductionion in the physiological content of calcium. Gel zymography demonstrated that calcium reduction did notinhibited significantly the Ca2+-dependent metalloprotease activities. In particular, high levels of a 90-85 kDa metalloprotease appeared and persisted during the development of Mn-exposed embryos. Taken together, these results explain the ability of Mn to interfere with calcium transport/accumulation into embryos and suggest that skeleton growth is highly dependent on calcium signaling. The use of Mn-exposed embryos as a new model to study skeleton signalling pathways is proposed