Bonaventura R et al. (2022), Gene Expression Analysis of the Stress Response...

ECB-ART-50267

Toxics 2022 Jun 14;106:. doi: 10.3390/toxics10060325.

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Gene Expression Analysis of the Stress Response to Lithium, Nickel, and Zinc in Paracentrotus lividus Embryos.

Bonaventura R , Costa C , Deidda I , Zito F , Russo R .

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Many anthropogenic pollutants such as metals are discharged into the marine environment through modern sources. Among these, lithium (Li), nickel (Ni), and zinc (Zn) can interfere with biological processes in many organisms when their concentration rises. These metals are toxic to sea urchin embryos, affecting their development. Indeed, animal/vegetal and dorso/ventral embryonic axes are differently perturbed: Li is a vegetalizing agent, Ni can disrupt dorso-ventral axis, Zn can be animalizing. To address the molecular response adopted by embryos to cope with these metals or involved in the gene networks regulating embryogenesis, and to detect new biomarkers for evaluating hazards in polluted environments in a well-known in vivo model, we applied a high-throughput screening approach to sea urchin embryos. After fertilization, Paracentrotus lividus embryos were exposed to Li, Ni, and Zn for 24/48 h. At both endpoints, RNAs were analyzed by NanoString nCounter technology. By in silico analyses, we selected a panel of 127 transcripts encoding for regulatory and structural proteins, ranked in categories: Apoptosis, Defense, Immune, Nervous, Development, and Biomineralization. The data analysis highlighted the dysregulation of many genes in a metal-dependent manner. A functional annotation analysis was performed by the KEEG Orthology database. This study provides a platform for research on metals biomarkers in sea urchins.

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	Figure 1. Morphologies of P. lividus embryos treated with Li, Ni, and Zn. (A) Schematic drawing of Li, Ni and Zn treatment experiments. Black line, hpf, in the upper part; the first vertical arrow indicates addition of 30 mM LiCl (Li), 0.5mM NiCl2 (Ni), 0.1mM ZnSO4 (Zn) at 0.5 hpf. Periods of treatment with Li, orange line; Ni, red line; Zn, yellow line. Untreated embryos (CTR), blue line. s/o indicates sampling (s) for NanoString nCounter gene expression assay and qPCR analyses and (o) microscopic observation. Embryos were observed at 24 (B–E) and 48 (F–I) hours post-fertilization (hpf). Untreated embryos: (B) control gastrula; (F) control pluteus. (C,G) Li-embryos; (D,H) Ni-embryos; (E,I) Zn-embryos. Blue ellipses in (D,H) indicate short archenteron. Black arrows in (D,E) indicate cells grouped inside the blastocoelic cavity. Red arrows in (F,G) indicate pigment cells. Schemes of embryos at 48 hpf: control pluteus, CTR (J); Li-embryo, Li (K); Ni-embryo, Ni (L); Zn-embryo, Zn (M). Animal/Vegetal (A/V), Dorso/Ventral (D/V) axes are indicated in the scheme of pluteus embryo. (N–Q) Anti-Sox2 antibody (red); (R–U) Ab-295 (green); (V–Y) Merged images (yellow) of the relative Anti-Sox2 (N–Q) and Ab-295 (R–U) images, respectively. The image in the white frame in (N) indicates 1.4× magnification of the pluteus apex, where brightness and contrast have been increased. White arrows in (N–Q) indicate Sox2-positive cells. White ellipses in (R) indicate small central ganglionic structures. White arrows in (R–T) and (V–X) indicate ciliary band. Bar 20 μm for (B–I) and (N–Y).
	Figure 2. Heat map of the 127 genes analyzed following metal treatments. The heat map was generated using conditional formatting on Microsoft Excel. Genes are reported in alphabetical order. Violet, green, and light yellow indicate fold values <−2, >+2 and between −2 and +2, respectively; light blue indicates not quantifiable genes. Li, embryos treated with 30 mM LiCl; Ni, embryos treated with 0.5 mM NiCl2; Zn, embryos treated with 0.1 mM ZnSO4. Hpf, hours post-fertilization.
	Figure 3. Percentages of genes modulated by metal treatments. Percentages of genes modulated by Li (30 mM LiCl), Ni (0.5 mM NiCl2) and Zn (0.1 mM ZnSO4) treatments at 24 and 48 hpf (hours post-fertilization). The fill colors indicate the upregulated genes in green (>+2), downregulated genes in violet (<−2), not responsive in light yellow (between −2 and +2), and not quantifiable (nq) in light blue.
	Figure 4. Apoptosis, Defense, Immune, and Nervous genes are modulated by Li, Ni, and Zn treatments. The grey boxes indicate the threshold for insignificant changes following Li, Ni, and Zn treatments, i.e., fold values between −2 and + 2 at 24 (A) and 48 (B) hours post-fertilization (hpf). Symbols in the plot at 24 hpf (A): light blue rhombus, Li-embryos; orange square, Ni-embryos; light green triangle, Zn-embryos. Symbols in the plot at 48 hpf (B): blue rhombus, Li-embryos; red square Ni-embryos; green triangle, Zn-embryos. Ap, Apoptosis genes; Def, Defense genes; Imm, Immune genes; Ner, Nervous genes.
	Figure 5. Development genes modulated by Li, Ni, and Zn treatments. The grey boxes indicate the threshold for insignificant changes following Li, Ni, and Zn treatments, i.e., fold values between −2 and + 2 at 24 (A) and 48 (B) hours post-fertilization (hpf). Symbols in the plot at 24 hpf (A): light blue rhombus, Li-embryos; orange square, Ni-embryos; light green triangle, Zn-embryos. Symbols in the plot at 48 hpf (B): blue rhombus, Li-embryos; red square Ni-embryos; green triangle, Zn-embryos. Some Development genes were organized considering their dorsal and ventral ectoderm expression. Asterisks (*) indicate genes also expressed in other territories.
	Figure 6. Development and Biomineralization genes with endomesoderm and mesoderm (primary mesenchyme cells, PMCs) expression modulated by Li, Ni, and Zn treatments. The grey boxes indicate the threshold for insignificant changes following Li, Ni, and Zn treatments, i.e., fold values between −2 and + 2, at 24 (A) and 48 (B) hours post-fertilization (hpf). Symbols in the plot at 24 hpf (A): light blue rhombus, Li-embryos; orange square, Ni-embryos; light green triangle, Zn-embryos. Symbols in the plot at 48 hpf (B): blue rhombus, Li-embryos; red square Ni-embryos; green triangle, Zn-embryos. The Development genes here considered have endomesoderm expression. Biomineralization genes, mainly expressed by the PMCs, were organized taking into consideration their functions, i.e., transcription factors (TFs), signaling molecules (signaling), and skeleton structures (skeleton). Asterisks (*) indicate genes also expressed in other territories.
	Figure 7. Comparison of qPCR data of gene transcription levels in Li-, Ni-, and Zn-embryos with NanoString data. Pl-alx1, Pl-can, Pl-gal8, Pl-jun, Pl-mt, and Pl-sox9 mRNA levels were analyzed compared to control embryos using the endogenous gene Pl-Z12-1 for normalization. The gray boxes indicate the threshold for insignificant changes following Li, Ni, and Zn treatments, i.e., fold values between −2 and + 2, at 24 (A) and 48 (B) hours post-fertilization (hpf). Symbols: NanoS, NanoString data, blue bars; qPCR data, red bars; Li, Li-embryos; Ni, Ni-embryos, Zn, Zn-embryos. The light blue triangles indicate not quantifiable values. Each bar of qPCR data represents the mean of three independent experiments of qPCR ± SD. QPCR mean values were significantly different according to the one-way ANOVA (p < 0.05), followed by the Tukey’s test. The asterisks (*) indicate statistically not significant variations to the relative control.
	Figure 8. Venn diagrams reporting the numbers of modulated genes in different subgroups, i.e., in Li, Ni, Zn, Li/Ni, Li/Zn, Ni/Zn, and Li/Ni/Zn. Modulated genes at 24 (A) and 48 (B) hours post-fertilization (hpf). The table below reports the numbers of down- and up-regulated genes in each group.

References [+] :

Andrikou, Myogenesis in the sea urchin embryo: the molecular fingerprint of the myoblast precursors. 2013, Pubmed, Echinobase