XB-ART-57622
Antioxidants (Basel)
December 12, 2020;
9
(12):
Xenopus gpx3 Mediates Posterior Development by Regulating Cell Death during Embryogenesis.
Abstract
Glutathione peroxidase 3 (GPx3) belongs to the glutathione peroxidase family of selenoproteins and is a key antioxidant enzyme in multicellular organisms against oxidative damage. Downregulation of GPx3 affects tumor progression and metastasis and is associated with
liver and
heart disease. However, the physiological significance of GPx3 in vertebrate embryonic development remains poorly understood. The current study aimed to investigate the functional roles of gpx3 during embryogenesis. To this end, we determined gpx3''s spatiotemporal expression using Xenopus laevis as a model organism. Using reverse transcription polymerase chain reaction (RT-PCR), we demonstrated the zygotic nature of this gene. Interestingly, the expression of gpx3 enhanced during the
tailbud stage of development, and whole mount in situ hybridization (WISH) analysis revealed gpx3 localization in prospective
tail region of developing
embryo. gpx3 knockdown using antisense morpholino oligonucleotides (MOs) resulted in short post-anal tails, and these malformed tails were significantly rescued by glutathione peroxidase mimic ebselen. The gene expression analysis indicated that gpx3 knockdown significantly altered the expression of genes associated with Wnt,
Notch, and
bone morphogenetic protein (BMP) signaling pathways involved in
tailbud development. Moreover, RNA sequencing identified that gpx3 plays a role in regulation of cell death in the developing
embryo. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and phospho-histone 3 (PH3) staining confirmed the association of gpx3 knockdown with increased cell death and decreased cell proliferation in
tail region of developing embryos, establishing the involvement of gpx3 in
tailbud development by regulating the cell death. Furthermore, these findings are inter-related with increased reactive oxygen species (ROS) levels in gpx3 knockdown embryos, as measured by using a redox-sensitive fluorescent probe HyPer. Taken together, our results suggest that gpx3 plays a critical role in
posterior embryonic development by regulating cell death and proliferation during vertebrate embryogenesis.
PubMed ID:
33322741
PMC ID:
PMC7764483
Article link:
Antioxidants (Basel)
Grant support:
[+]
2020R1A4A1018280, 2018R1A6A1A03025810 National Research Foundation of Korea, 2020R1F1A1048235 National Research Foundation of Korea
Genes referenced:
fgf8
gabpa
not
notch1
tbx6
tbxt
wnt3a
wnt5a
wnt5b
Article Images:
[+] show captions
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Figure 1. gpx3 is required for tailbud development during Xenopus embryonic development. (A) RT-PCR analysis indicated the zygotic gpx3 gene expression. gpx3 expression started at the gastrula stage of development (St.12) and continued until later stages (St.40). The highest expression levels were observed at the tailbud stage (St.20-25). Ornithine decarboxylase (odc) was used as an internal control. (B) Whole mount in situ hybridization (WISH) analysis demonstrated gpx3 expression at the prospective tailbud region (St.23) and exhibited its localization in the developing embryo tail region (St.31). (C) gpx3 morpholino oligonucleotides (MO) microinjection into the 4-cell stage embryo ventral side resulted in short post-anal tails in gpx3 morphant embryos compared to control MO-injected embryos. Malformed phenotypes induced by gpx3 knockdown were effectively rescued by ebselen, a GPx mimic. (D) Embryo phenotype quantification revealed that more than 90% of the injected embryos with gpx3 MO developed short post-anal tails compared to control embryos. The short tail phenotypes were 50% recovered in ebselen-treated embryos. * p <0.05, **** p <0.0001.
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Figure 2. gpx3 knockdown perturbed Wnt, Notch, and FGF signaling gene expression but did not affect the early tailbud gene expression. (A,B) gpx3 MO was injected into the ventral regions of 4-cell stage embryos, and RT-PCR was used to determine wnt3a, wnt5a, wnt5b, notch1, fgf8, and Xtbx6 gene expression after removing the anterior part of embryos. wnt3a, wnt5b, notch1, and fgf8 expressions were significantly reduced in gpx3 MO-injected embryos. * p <0.05; ** p <0.001; *** p <0.0001; ns, not significant. (C) WISH analysis of gpx3 morphant embryos using Xbra and Xnot2 (chordoneural hinge and posterior wall markers). gpx3 knockdown did not affect the early tailbud gene expression. (D) RT-PCR of Xbra and Xnot2 relative expression revealed no significant differences between the control and gpx3 MO-injected embryos.
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Figure 3. Perturbed gpx3 leads to increased reactive oxygen species (ROS) levels. Embryos were injected with 10 ng of HyPer mRNA at the 1-cell stage. MOs were injected into the ventral regions at the 4-cell stage. (A) gpx3 MO-injected embryos show increased HyPer fluorescent compared to the control. (B) HyPer fluorescence intensity quantification using ImageJ. * p <0.05.
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Figure 4. Effects of gpx3 knockdown on the Xenopus embryo transcriptome. (A) Heat map showing significant differences between control and gpx3 MO-injected embryos. (B) Transcriptome enrichment analysis using PANTHER indicated that cell death processes are significantly upregulated in gpx3-depleted embryos. (C) Gene ontology (GO-terms) and genes also indicated that genes associated with apoptosis and cell-death-related pathways were significantly affected by gpx3 depletion. (D) The expression of Nrf2 and its putative targets were not affected by gpx3 knockdown.
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Figure 5. gpx3 suppression activates apoptosis and inhibits cell proliferation in gpx3 morphants. (A) The expression and number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells were higher in gpx3 knockdown embryos compared to control embryos. (B) Relative TUNEL expression, indicating a significant increase in apoptotic cells in gpx3 morphants. ** p <0.001. (C) gpx3 loss resulted in reduced phospho-histone 3 (PH3)-positive cells in the tail region of gpx3 knockdown embryos. PH3-positive cells are indicated by the color purple, whereas β-gal staining is indicated by the color blue. (D) The relative PH3 expression showed cell proliferation was significantly reduced by gpx3 knockdown. * p <0.05.
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