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

Annalisa Pinsino 0 1 Maria Carmela Roccheri 1 Caterina Costa 0 Valeria Matranga 0 0 Istituto di Biomedicina e Immunologia Molecolare ''Alberto Monroy,'' Consiglio Nazionale delle Ricerche , 90146 Palermo , Italy 1 Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari, Universita` di Palermo , 90128 Palermo , Italy The Author 2011. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: - fast morphogenesis, and biochemical similarity to vertebrates. In the sea urchin embryo, development is controlled by gene regulatory networks (GRNs) that specify cell fates at the appropriate time and space. Founder cells and their three germ layers progenies are the basic units where regulatory information is localized during cleavage (Angerer and Angerer, 2007; Livingston and Wilt, 1990). The primary mesenchyme cells (PMCs) founders appear at fourth cleavage and become autonomously specified by b-catenin-induced transcriptional activation. Later, b-catenin is required for the development of all endo-mesoderm territories (Logan et al., 1999). Finally, cell fates are fully specified by the blastula-early gastrula stage of development, when cells have begun to express particular sets of territory-specific genes (Davidson et al., 1998). Although maternal determinants are required for founder cells specification during development, interactions between the PMCs and external cues derived from the ectoderm specify many phases of the skeleton formation and patterning (skeletogenesis) (Armstrong et al., 1993; Ettensohn and Malinda, 1993; Guss and Ettensohn, 1997; Zito et al., 1998). Skeletogenesis begins with the accumulation and secretion of the biomineral within a privileged extracellular space enshrouded by the fused PMCs filopodial processes (Dubois and Chen, 1989; Wilt, 2002, 2005). PMCs utilize spatial and temporal signals to organize the proper animal-vegetal and oral-aboral position, formation, and orientation of the two initial triradiate skeletal spicules (Duloquin et al., 2007; R ottinger et al., 2008; Zito et al., 2003). The two spicule rudiments elongate and branch in a threedimensional endoskeleton composed of magnesian calcite and spicule matrix proteins (Killian and Wilt, 1996, 2008). Many of the proteins involved in biomineralization are members of small families of coordinately expressed genes clustered in the genome, including the spicule matrix proteins SM30, SM50, and the cell surface protein MSP130 (Livingston et al., 2006). At gastrulation, PMCs transmit an inhibitory signal to the secondary mesenchyme cells (SMCs) preventing their differentiation into skeletogenic mesenchyme, thus promoting the 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. K e y Wo r d s : m e t a l s ; a q u a t i c t o x i c o l o g y ; e m b r y o ; biomineralization; development. Sea urchins provide an attractive and tractable embryonic model for exploring the mechanisms used for successful development as it produces large numbers of transparent embryos exhibiting rapid cell divisions during cleavage stages, production of a variety of differentiated mesodermal cells suggesting that SMCs function as multipotent stem cells (Kiyomoto et al., 2007; Zito and Matranga, 2009). It has been widely demonstrated that extracellular signal-regulated kinase (ERK)-mediated signaling controls the expression of several regulatory genes, which participate in the specification and differentiation of mesenchyme cells (Ettensohn, 2009; Livingston et al., 2006; Ro ttinger et al., 2004). During development, ERK is activated in a spatial-temporal manner: its activated form is localized in prospective PMCs and SMCs during their epithelial-mesenchyme transition and it is downregulated immediately after their transition. Manganese is an essential mineral nutrient needed for proper fetal development and other important aspects of metabolism (Wood, 2009). However, Mn excess can have a potent neurotoxic effect, especially in infants (Chung et al., 2011; Santamaria, 2008). Although environmental toxicology studies described some of the adverse effects of high Mn exposure in humans, little is known about the effects of Mn toxicity on fetal and newborn development (Vigeh et al., 2008; Zota et al., 2009). Interestingly, a number of reports have shown that Mn and Ca trafficking, recruitment, and storage are regulated in mammalian cells by the same ion pumps and in the same intracellular compartments (Van Baelen et al., 2004; Vanoevelen et al., 2005). A great variety of developmental processes such as egg activation and fertilization, cellular cleavage, neuronal development, and cell death are known to be dependent on the dynamic release of Ca ions (Roux et al., 2006; Santella et al., 2004; Slusarski and Pelegri, 2007; Whitaker, 2006; Yazaki, 2001). A particularly important emerging concept is that Ca can trigger several specific cellular responses by changes in the amplitude, frequency, and duration of its intracellular oscillations. A few studies have shown that Ca oscillation (...truncated)