Effects of cadmium exposure on sea urchin development assessed by SSH and RT-qPCR: metallothionein genes and their differential induction

Abstract

In order to study the defense strategies activated by Paracentrotus lividus embryos in response to sub-lethal doses of CdCl2, we compared the induced transcripts to that of control embryos by suppression subtractive hybridization technique. We isolated five metallothionein (MT) cDNAs and other genes related to detoxification, to signaling pathway components, to oxidative, reductive and conjugative biotransformation, to RNA maturation and protein synthesis. RT-qPCR analysis revealed that two of the five P. lividus MT (PlMT7 and PlMT8) genes appeared to be constitutively expressed and upregulated following cadmium treatment, whereas the other three genes (PlMT4, PlMT5, PlMT6) are specifically switched-on in response to cadmium treatment. Moreover, we found that this transcriptional induction is concentration dependent and that the cadmium concentration threshold for the gene activation is distinct for every gene. RT-qPCR experiments showed in fact that, among induced genes, PlMT5 gene is activated at a very low cadmium concentration (0.1 μM) whereas PlMT4 and PlMT6 are activated at intermediate doses (1-10 μM). Differently, PlMT7 and PlMT8 genes increase significantly their expression only in embryos treated with the highest dose (100 μM CdCl2). We found also that, in response to a lethal dose of cadmium (1 μM), only PlMT5 and PlMT6 mRNA levels increased further. These data suggest a hierarchical and orchestrated response of the P. lividus embryo to overcome differential environmental stressors that could interfere with a normal development.

Tissue-specificity and phylogenetics of Pl-MT mRNA during Paracentrotus lividus embryogenesis

“…In particular, they suggested a possible role for MT as a reservoir of zinc required for DNA synthesis during normal development. Actually, a direct link has been shown between the induction of Pl-MT mRNAs and the exposure to heavy metal accumulation under stress conditions (Ragusa et al, 2012;Russo et al, 2003). On the contrary, we need to further examine the MT mRNA turnover under physiological conditions and clarify if there is any relationship with the turnover of essential metals during P. lividus development.…”

“…Actually, when embryos were continuously cultured in the presence of CdCl 2 from fertilization onwards, a significant increase of Pl-MT mRNA expression has been detected as early as 6 h after exposure, corresponding to the cleavage stage (Russo et al, 2003). Similarly, a significant increase in PlMT8 expression has been described in embryos treated with high doses of CdCl 2 (Ragusa et al, 2012). Thus, the constitutive and inducible expression patterns observed suggest a dual role for Pl-MT: as a housekeeping protein in the homeostasis of heavy metal ions during embryonic development and as a defense protein against the accumulation of xenobiotic metals.…”

“…In Paracentrotus lividus embryos, the first complete MT cDNA had been obtained in our lab and referred to as Pl-MT (Russo et al, 2003). Very recently, Ragusa et al (2012) reported the isolation of five different MT cDNAs from P. lividus embryo. Two of the five MT genes (PlMT7 and PlMT8) are constitutively expressed and upregulated in response to cadmium treatment, whereas the other three genes (PlMT4, PlMT5 and PlMT6) appear to be specifically switched-on after cadmium treatment (Ragusa et al, 2012).…”

“…Very recently, Ragusa et al (2012) reported the isolation of five different MT cDNAs from P. lividus embryo. Two of the five MT genes (PlMT7 and PlMT8) are constitutively expressed and upregulated in response to cadmium treatment, whereas the other three genes (PlMT4, PlMT5 and PlMT6) appear to be specifically switched-on after cadmium treatment (Ragusa et al, 2012). Moreover, by nucleotide sequence alignment performed by BLAST, the authors showed a high identity between Pl-MT (Russo et al, 2003) and PlMT8, one of the constitutively expressed genes.…”

Autophagy as a defense strategy against stress: focus on Paracentrotus lividus sea urchin embryos exposed to cadmium

“…1). Metallothionein expression (Pl-MT gene) Russo et al (2003) Blastula (15 h), gastrula (24 h) Synthesis of a specific set of HSPs Roccheri et al (2004) Blastula (15 h Gastrula (24 h) Autophagy (by LC3-II immunodetection) Chiarelli and Roccheri (2012) Gastrula (24 h) Apoptosis-autophagy interplay (by TUNEL, caspase-3, and LC3-II immunodetection, AO staining 100 μM Pluteus (38 h) Apoptosis (by TUNEL assay and caspase-3 immunodetection) Agnello et al (2006) Pluteus (38 h) Autophagy (by AO staining) Chiarelli and Roccheri (2014) 1 μM, 1 nM, 1 pM Larva (10-30 days) Apoptosis (by TUNEL assay) Filosto et al (2008) 0.1-100 μM Gastrula (24 h), prisma (30 h) Metallothionein expression (PlMT4, PlMT5, PlMT6, PlMT7, and PlMT8 genes) Ragusa et al (2013) 0.5-30 μM 2-cell, 8-cell, early blastula, swimming blastula, prisma, pluteus…”