Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari, Università di Palermo, 90128 Palermo, Italy.
Toxicol Sci. 2011 Sep;123(1):217-30. doi: 10.1093/toxsci/kfr152. Epub 2011 Jun 9.
Manganese (Mn) has been associated with embryo toxicity as it impairs differentiation of neural and skeletogenic cells in vertebrates. Nevertheless, information on the mechanisms operating at the cellular level remains scant. We took advantage of an amenable embryonic model to investigate the effects of Mn in biomineral formation. Sea urchin (Paracentrotus lividus) embryos were exposed to Mn from fertilization, harvested at different developmental stages, and analyzed for their content in calcium (Ca), expression of skeletogenic genes, localization of germ layer markers, and activation of the extracellular signal-regulated kinase (ERK). By optical and immunofluorescence microscopy, we found that Mn exposure produced embryos with no skeleton, by preventing the deposition of the triradiate calcitic spicules usually produced only by specialized mesoderm cells. On the contrary, ectoderm and endoderm differentiation was not impaired. Endogenous Ca content in whole embryos and its localization in Golgi regions of skeletogenic cells was strongly reduced, as measured by atomic absorption spectrometry and in vivo calcein labeling. Spicule-lacking embryos showed persistent ERK activation by immunocytochemistry and immunoblotting, contrary to the physiological oscillations observed in normal embryos. The expression of the skeletogenic genes, Pl-msp130 and Pl-sm30, was also differentially affected if compared with controls. Here, we showed for the first time the ability of Mn to interfere with Ca uptake and internalization into skeletogenic cells and demonstrate that Ca content regulates ERK activation/inactivation during sea urchin embryo morphogenesis. The use of Mn-exposed sea urchin embryos as a new model to study signaling pathways occurring during skeletogenesis will provide new insights into the mechanisms involved in Mn embryo toxicity and underlie the role of calcium in the biomineralization process in vertebrates.
锰（Mn）与胚胎毒性有关，因为它会损害脊椎动物的神经和骨骼细胞的分化。然而，关于细胞水平上的作用机制的信息仍然很少。我们利用一种易于处理的胚胎模型来研究 Mn 在生物矿化形成中的作用。将海胆（Paracentrotus lividus）胚胎从受精开始暴露于 Mn，在不同的发育阶段收获，并分析其钙（Ca）含量、骨骼基因表达、胚层标记物的定位以及细胞外信号调节激酶（ERK）的激活情况。通过光学和免疫荧光显微镜观察，我们发现 Mn 暴露会阻止通常仅由专门的中胚层细胞产生的三辐射碳酸钙刺的沉积，从而导致胚胎没有骨骼。相反，外胚层和内胚层分化不受影响。通过原子吸收光谱法和体内 calcein 标记法测量，整个胚胎的内源性 Ca 含量及其在骨骼细胞高尔基体区域的定位均明显减少。免疫细胞化学和免疫印迹显示，无刺状的胚胎中 ERK 持续激活，与正常胚胎中观察到的生理振荡相反。如果与对照相比，骨骼基因 Pl-msp130 和 Pl-sm30 的表达也受到不同程度的影响。在这里，我们首次展示了 Mn 干扰骨骼细胞摄取和内化 Ca 的能力，并证明 Ca 含量调节 ERK 在海胆胚胎形态发生过程中的激活/失活。利用暴露于 Mn 的海胆胚胎作为研究骨骼发生过程中发生的信号通路的新模型，将为 Mn 胚胎毒性的作用机制提供新的见解，并阐明钙在脊椎动物生物矿化过程中的作用。
