Manganese Interferes with Calcium, Perturbs ERK Signaling, and Produces Embryos with No Skeleton

Abstract

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.

Manganese overload affects p38 MAPK phosphorylation and metalloproteinase activity during sea urchin embryonic development

“…The MMPs activity detected in Mn-exposed embryos, in the presence or absence of EDTA, was very similar, suggesting that the proteolytic cascade inhibition could be due to a cellular calcium deficiency, since Mn is competing with calcium uptake, as demonstrated previously (Pinsino et al, 2011(Pinsino et al, , 2012. In control embryos, this proteolytic cascade (i.e.…”

“…Rather, we observed a concentration-dependent increase in the number of morphological abnormalities and in the levels of the hsc70 and hsc60 proteins (Pinsino et al, 2010). In addition, we found that the exposure to the highest Mn concentration tested (61.6 mg l À1 or 1.12 mM) prevents skeleton growth which result in spicule-lacking embryos, interferes with calcium uptake and internalization into cells, and causes remarkable consequences in skeletogenic gene expression and ERK mediated signaling pathway (Pinsino et al, 2011). The MAPK proteins are members of a large group of proteins that allow cells to perceive changes in the intraand extracellular environment and respond to them appropriately.…”

“…), severely inhibited skeleton formation in totality of the embryos (100%), we extended our studies concerning the effects of Mn toxicity on sea urchin development at the molecular (Pinsino et al, 2011) and biochemical level (this study). In the sea urchin embryo, the development of the endoskeleton is an essential step for constructing the framework of the "body"; the absence of embryonic skeleton greatly influences the fitness of the resulting larvae reducing their feeding capability.…”

Effects of exposure to gadolinium on the development of geographically and phylogenetically distant sea urchins species

“…A similar approach was used for the other three species under investigation, with final Gd concentrations used ranging from 1 nm to 200 mM. For each experiment 50 embryos from three different wells were sampled at the two developmental stages, gastrula (24 h post fertilization-hpf) and pluteus (48 hpf), as in previous studies (Bonaventura et al, 2005;Pinsino et al, 2011). In some experiments, a total number of 100 or 300 embryos were sampled (see Table 1).…”

“…For instance, exposure to toxic elements (e.g. cadmium and manganese), UVB and X rays inhibits skeletogenesis (Filosto et al, 2008;Pinsino et al, 2011;Bonaventura et al, 2005;Matranga et al, 2010). The sea urchin larval skeleton is produced by the primary mesenchyme cells (PMCs) that migrate into the blastocoel during gastrulation, forming two ventrolateral clusters that secrete the spicule rudiment (Killian and Wilt, 2008;Matranga et al, 2011).…”