In the sea urchin embryos, skeleton is specified by interactions between PMCsand patterning cues derived from the ectoderm. PMCs use spatial andtemporal information to organize the proper animal-vegetal and oral-aboralposition and orientation of the two tri-radiate skeletal spicules. Manyexperiments have demonstrated that exposure to metals, such as lithium, zincand nickel, can disrupt skeleton patterning information. Here, we haveinvestigated the effects of manganese (Mn) on Paracentrotus lividus embryodevelopment. We found that Mn exposure prevents skeleton growth producingspicule-lacking embryos. Normal skeleton growth was partially rescued afterMn removal from the culture. To determine the effects of Mn exposure on thedifferentiation of the three germ layers, Mn-exposed embryos wereimmunostained 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 inMn-exposed skeleton-lacking embryos at the appropriate time and in thecorrect position. By in situ hybridization we analyzed the expression of threegenes expressed during PMCs differentiation in Mn-exposed embryos, SM50,SM30 and msp130, encoding two spicule matrix proteins and the cell surfaceprotein detected by 1D5 Mab, respectively. Results showed that: i) SM50expression was largely normal; ii) SM30 expression was severely reduced andiii) msp130 expression was not down-regulated during development. Activationof p42-44 and p38 MapKs was analyzed by Western blotting in Mn-exposedembryos during their development. We found a persistent phosphorylatedstate of both MapKs, as proteins levels were only partially modulated duringdevelopment of Mn-exposed embryos. Results from fluorescent labeling ofintracellular calcium, obtained by calcein labelling, and the amount of calciumconcentration, determined by atomic absorption spectrophotometer in thesame specimens, showed a reductionion in the physiological content ofcalcium. Gel zymography demonstrated that calcium reduction did notinhibited significantly the Ca2+-dependent metalloprotease activities. Inparticular, high levels of a 90-85 kDa metalloprotease appeared and persistedduring the development of Mn-exposed embryos. Taken together, theseresults explain the ability of Mn to interfere with calciumtransport/accumulation into embryos and suggest that skeleton growth ishighly dependent on calcium signaling. The use of Mn-exposed embryos as anew model to study skeleton signalling pathways is proposed.
|Numero di pagine||2|
|Stato di pubblicazione||Published - 2009|