Lee H et al. (2021), Physiological Functions of Thiol Peroxidases (G...

XB-ART-58551

Antioxidants (Basel) 2021 Oct 17;1010:. doi: 10.3390/antiox10101636.

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Physiological Functions of Thiol Peroxidases (Gpx1 and Prdx2) during Xenopus laevis Embryonic Development.

Lee NY , Kim Y , Choi HS , Ismail T , Ryu HY , Cho DH , Ryoo ZY , Lee DS , Kwon TK , Park TJ , Kwon T .

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Glutathione peroxidase 1 (Gpx1) and peroxiredoxin 2 (Prdx2) belong to the thiol peroxidase family of antioxidants, and have been studied for their antioxidant functions and roles in cancers. However, the physiological significance of Gpx1 and Prdx2 during vertebrate embryogenesis are lacking. Currently, we investigated the functional roles of Gpx1 and Prdx2 during vertebrate embryogenesis using Xenopus laevis as a vertebrate model. Our investigations revealed the zygotic nature of gpx1 having its localization in the eye region of developing embryos, whereas prdx2 exhibited a maternal nature and were localized in embryonic ventral blood islands. Furthermore, the gpx1-morphants exhibited malformed eyes with incompletely detached lenses. However, the depletion of prdx2 has not established its involvement with embryogenesis. A molecular analysis of gpx1-depleted embryos revealed the perturbed expression of a cryba1-lens-specific marker and also exhibited reactive oxygen species (ROS) accumulation in the eye regions of gpx1-morphants. Additionally, transcriptomics analysis of gpx1-knockout embryos demonstrated the involvement of Wnt, cadherin, and integrin signaling pathways in the development of malformed eyes. Conclusively, our findings indicate the association of gpx1 with a complex network of embryonic developmental pathways and ROS responses, but detailed investigation is a prerequisite in order to pinpoint the mechanistic details of these interactions.

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Species referenced: Xenopus laevis
Genes referenced: cryba1

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	Figure 1. Spatiotemporal expression pattern of gpx1 and prdx2 during Xenopus embryogenesis. (A) The transcriptase-polymerase chain reaction (RT-PCR) analysis indicated the zygotic nature of gpx1 during Xenopus embryonic development. The temporal expression of gpx1 started at NF. St. 30 of embryogenesis and proceeded until NF. St. 40. The highest expression level was observed at NF. St. 35 of Xenopus embryogenesis. Ornithine decarboxylase (odc) was used as an internal loading control. (B) The general scheme for Xenopus eye development in the tadpole stage indicates eye vesicle formation at NF. St. 25, followed by lens induction at NF. St. 30., while lens detachment begins at NF. St. 35, leading to cornea formation at NF. St 40 of Xenopus embryonic development. (C) The spatial expression of gpx1 as analyzed by whole-mount in situ hybridization (WISH) indicated its specific expression in the lens after eye vesicle formation at NF. St 30 embryonic development with an increased expression level in the lens region at NF. St. 35 and it proceeded until NF. St. 40 of Xenopus embryogenesis. (D) RT-PCR analysis revealed the maternal nature of prdx2 during Xenopus embryogenesis. The highest level of prdx2 expression was observed at the late neurula and tailbud stages of embryogenesis. Ornithine decarboxylase (odc) was used as the internal loading control. (E) WISH analysis indicated the localization of prdx2 in embryonic VBIs during Xenopus embryonic development.
	Figure 2. gpx1 loss-of-function results in malformed eyes with incompletely detached lenses, while prdx2 morphant embryos have no phenotypic malformations. (A) Two types of gpx1 morpholino oligonucleotides (MOs), i.e., translational blocking MO (gpx1 MO; 20 ng) and splicing blocking MO (gpx1 spMO; 5 ng), were microinjected into animal poles of two-cell stage Xenopus embryos, and then the embryos were fixed at NF. St. 40. Knockdown of gpx1 in both groups of MOs resulted in malformed eyes, as shown in the enlarged images. Additionally, the cross-sectional analysis indicated the incompletely detached lens in both groups of gpx1 morphants. (B) The statistical analysis of gpx1 morphants indicated that more than 70% of the embryos exhibited malformed eyes in the case of gpx1 MO microinjection, while more than 50% of embryos showed eye malformations in the case of gpx1 spMO injection compared with the control embryos. p ≤ 0.01, * p ≤ 0.001. (C) Rescue experiments were conducted by transferring the gpx1 morpholino injected embryos to 0.5-μΜ ebselen, a Gpx mimic, and then fixed at NF. St. 40. The eye malformations observed in both groups of gpx1-depleted embryos were effectively recovered after ebselen treatment. (D) A graphical representation of rescued embryos showed that more than 30% of embryos recovered the malformed eyes observed due to gpx1 MO and gpx1 spMO microinjection after ebselen treatment. * p ≤ 0.05 p ≤ 0.01, * p ≤ 0.001, **** p ≤ 0.0001. (E) prdx2 MO (40 ng) and wildtype prdx2 mRNA (1 ng) were microinjected into the two cell stage developing embryos and then the embryos were fixed at NF. St. 40. The embryos developed normally after prdx2 MO injection and there were no phenotypic differences between prdx2 morphant embryos and embryo injected with wildtype prdx2 mRNA. Both group of embryos were morphologically similar to the control MO injected embryos. (F) Xenopus embryos were injected with prdx2 MO and were processed for WISH analysis using blood cell markers, i.e., hba3 and mpo. The knockdown of prdx2 did not affect the expression pattern of the blood cell-specific markers.
	Figure 3. Loss of gpx1 results in a perturbed expression of the lens-specific marker and an accumulation of ROS in developing eyes during Xenopus embryonic development. Here, (A) 20-ng gpx1 MO and 5-ng gpx1 spMO were microinjected into the animal pole of two-cell stage embryos, and the embryos were processed for WISH analysis using the lens-specific marker cryba1. The expression of cryba1 was remarkably reduced in both groups of gpx1-morphant embryos. Moreover, the downregulated expression of cryba1 was effectively rescued in gpx1 morphants after ebselen treatment. (B) A graphical representation of gpx1 MO-injected embryos revealed that gpx1-morphant embryos exhibited a lens diameter of 70 μm compared with >80 μm in the control embryos, and it was effectively recovered to 80 μm in the gpx1-depleted embryos after ebselen treatment. ** p ≤ 0.0001. (C) Statistical analysis of gpx1 spMO-injected embryos indicated that gpx1-depleted embryos showed a lens diameter of approximately 75 μm as compared with >80 μm in the control embryos. The smaller size of the lens was effectively rescued to 80 μm after ebselen treatment in the gpx1 spMO-injected embryos. * p ≤ 0.001, ** p ≤ 0.0001. (D) Both gpx1 MOs with HyPer mRNA (10 ng) were injected into the D.1.2. blastomeres of 16 cell stage Xenopus embryos and the gpx1-depleted embryos exhibited increased fluorescence intensity in the eye regions compared with the control embryos. (E) HyPer fluorescence intensity quantification clearly showed significant intense fluorescence in the gpx1 MO and gpx1 spMO injected embryos as compared with the control embryos. ** p ≤ 0.0001.
	Figure 4. gpx1 depletion activates cell apoptosis and inhibits cell proliferation, as well as affects cell migration during Xenopus embryogenesis. (A) The depletion of gpx1 increased TUNEL-positive cells in the eye region of developing gpx1 morphants compared with the control embryos. n.s. > 0.05, * p ≤ 0.001, ** p ≤ 0.0001. (B) gpx1 MO was coinjected with β-galactosidase mRNA into the D.1.2 blastomere of 16-cell stage Xenopus embryos. The staining showed that the cells were concentrated in the midline of the developing gpx1-depleted embryos compared with the widely distributed cells in the control embryos. The migration index was considerably reduced after gpx1 MO injection. * p ≤ 0.05 (C) The injection of gpx1 spMO into the D.1.2. blastomere also resulted in a cell concentration in the midline of developing embryos and decreased the migration index as compared with the control embryos. *** p ≤ 0.001.
	Figure 5. Transcriptomic analyses provide insight into the mechanism of eye development by gpx1 during Xenopus embryogenesis. (A) The heat map shows the remarkable difference between the control embryos and gpx1-morphant embryos. (B) Transcriptome enrichment analysis using PANTHER software revealed the upregulation of the biological pathways associated with the response to reactive oxygen species, oxygen transport, hydrogen peroxide, hydrogen peroxide catabolic process, and gas transport. (C) Transcriptomic analysis using PANTHER software demonstrated the downregulation of signaling pathways, i.e., Wnt, Cadherin, and integrin associated with eye development in gpx1-depleted embryos. (D) Genes associated with Wnt signaling (Wnt10b, Wnt 7b, and Wnt6) showed significantly low RPKM values in gpx1-depleted embryos compared with the control embryos. A similar case was observed for cadherin signaling-associated genes (CDH20, PCDH19, and PCDHGB5), showing considerably low RPKM values compared with the control embryos. Additionally, genes associated with eye development, such as RBP1, ALDH1A3, and VAX2, exhibited remarkably low RPKM values after gpx1 knockout compared with the control embryos.

References [+] :

Abouzeid, Mutations in ALDH1A3 represent a frequent cause of microphthalmia/anophthalmia in consanguineous families. 2014, Pubmed

	Figure 1. Spatiotemporal expression pattern of gpx1 and prdx2 during Xenopus embryogenesis. (A) The transcriptase-polymerase chain reaction (RT-PCR) analysis indicated the zygotic nature of gpx1 during Xenopus embryonic development. The temporal expression of gpx1 started at NF. St. 30 of embryogenesis and proceeded until NF. St. 40. The highest expression level was observed at NF. St. 35 of Xenopus embryogenesis. Ornithine decarboxylase (odc) was used as an internal loading control. (B) The general scheme for Xenopus eye development in the tadpole stage indicates eye vesicle formation at NF. St. 25, followed by lens induction at NF. St. 30., while lens detachment begins at NF. St. 35, leading to cornea formation at NF. St 40 of Xenopus embryonic development. (C) The spatial expression of gpx1 as analyzed by whole-mount in situ hybridization (WISH) indicated its specific expression in the lens after eye vesicle formation at NF. St 30 embryonic development with an increased expression level in the lens region at NF. St. 35 and it proceeded until NF. St. 40 of Xenopus embryogenesis. (D) RT-PCR analysis revealed the maternal nature of prdx2 during Xenopus embryogenesis. The highest level of prdx2 expression was observed at the late neurula and tailbud stages of embryogenesis. Ornithine decarboxylase (odc) was used as the internal loading control. (E) WISH analysis indicated the localization of prdx2 in embryonic VBIs during Xenopus embryonic development.
	Figure 2. gpx1 loss-of-function results in malformed eyes with incompletely detached lenses, while prdx2 morphant embryos have no phenotypic malformations. (A) Two types of gpx1 morpholino oligonucleotides (MOs), i.e., translational blocking MO (gpx1 MO; 20 ng) and splicing blocking MO (gpx1 spMO; 5 ng), were microinjected into animal poles of two-cell stage Xenopus embryos, and then the embryos were fixed at NF. St. 40. Knockdown of gpx1 in both groups of MOs resulted in malformed eyes, as shown in the enlarged images. Additionally, the cross-sectional analysis indicated the incompletely detached lens in both groups of gpx1 morphants. (B) The statistical analysis of gpx1 morphants indicated that more than 70% of the embryos exhibited malformed eyes in the case of gpx1 MO microinjection, while more than 50% of embryos showed eye malformations in the case of gpx1 spMO injection compared with the control embryos. p ≤ 0.01, * p ≤ 0.001. (C) Rescue experiments were conducted by transferring the gpx1 morpholino injected embryos to 0.5-μΜ ebselen, a Gpx mimic, and then fixed at NF. St. 40. The eye malformations observed in both groups of gpx1-depleted embryos were effectively recovered after ebselen treatment. (D) A graphical representation of rescued embryos showed that more than 30% of embryos recovered the malformed eyes observed due to gpx1 MO and gpx1 spMO microinjection after ebselen treatment. * p ≤ 0.05 p ≤ 0.01, * p ≤ 0.001, **** p ≤ 0.0001. (E) prdx2 MO (40 ng) and wildtype prdx2 mRNA (1 ng) were microinjected into the two cell stage developing embryos and then the embryos were fixed at NF. St. 40. The embryos developed normally after prdx2 MO injection and there were no phenotypic differences between prdx2 morphant embryos and embryo injected with wildtype prdx2 mRNA. Both group of embryos were morphologically similar to the control MO injected embryos. (F) Xenopus embryos were injected with prdx2 MO and were processed for WISH analysis using blood cell markers, i.e., hba3 and mpo. The knockdown of prdx2 did not affect the expression pattern of the blood cell-specific markers.
	Figure 3. Loss of gpx1 results in a perturbed expression of the lens-specific marker and an accumulation of ROS in developing eyes during Xenopus embryonic development. Here, (A) 20-ng gpx1 MO and 5-ng gpx1 spMO were microinjected into the animal pole of two-cell stage embryos, and the embryos were processed for WISH analysis using the lens-specific marker cryba1. The expression of cryba1 was remarkably reduced in both groups of gpx1-morphant embryos. Moreover, the downregulated expression of cryba1 was effectively rescued in gpx1 morphants after ebselen treatment. (B) A graphical representation of gpx1 MO-injected embryos revealed that gpx1-morphant embryos exhibited a lens diameter of 70 μm compared with >80 μm in the control embryos, and it was effectively recovered to 80 μm in the gpx1-depleted embryos after ebselen treatment. ** p ≤ 0.0001. (C) Statistical analysis of gpx1 spMO-injected embryos indicated that gpx1-depleted embryos showed a lens diameter of approximately 75 μm as compared with >80 μm in the control embryos. The smaller size of the lens was effectively rescued to 80 μm after ebselen treatment in the gpx1 spMO-injected embryos. * p ≤ 0.001, ** p ≤ 0.0001. (D) Both gpx1 MOs with HyPer mRNA (10 ng) were injected into the D.1.2. blastomeres of 16 cell stage Xenopus embryos and the gpx1-depleted embryos exhibited increased fluorescence intensity in the eye regions compared with the control embryos. (E) HyPer fluorescence intensity quantification clearly showed significant intense fluorescence in the gpx1 MO and gpx1 spMO injected embryos as compared with the control embryos. ** p ≤ 0.0001.
	Figure 4. gpx1 depletion activates cell apoptosis and inhibits cell proliferation, as well as affects cell migration during Xenopus embryogenesis. (A) The depletion of gpx1 increased TUNEL-positive cells in the eye region of developing gpx1 morphants compared with the control embryos. n.s. > 0.05, * p ≤ 0.001, ** p ≤ 0.0001. (B) gpx1 MO was coinjected with β-galactosidase mRNA into the D.1.2 blastomere of 16-cell stage Xenopus embryos. The staining showed that the cells were concentrated in the midline of the developing gpx1-depleted embryos compared with the widely distributed cells in the control embryos. The migration index was considerably reduced after gpx1 MO injection. * p ≤ 0.05 (C) The injection of gpx1 spMO into the D.1.2. blastomere also resulted in a cell concentration in the midline of developing embryos and decreased the migration index as compared with the control embryos. *** p ≤ 0.001.
	Figure 5. Transcriptomic analyses provide insight into the mechanism of eye development by gpx1 during Xenopus embryogenesis. (A) The heat map shows the remarkable difference between the control embryos and gpx1-morphant embryos. (B) Transcriptome enrichment analysis using PANTHER software revealed the upregulation of the biological pathways associated with the response to reactive oxygen species, oxygen transport, hydrogen peroxide, hydrogen peroxide catabolic process, and gas transport. (C) Transcriptomic analysis using PANTHER software demonstrated the downregulation of signaling pathways, i.e., Wnt, Cadherin, and integrin associated with eye development in gpx1-depleted embryos. (D) Genes associated with Wnt signaling (Wnt10b, Wnt 7b, and Wnt6) showed significantly low RPKM values in gpx1-depleted embryos compared with the control embryos. A similar case was observed for cadherin signaling-associated genes (CDH20, PCDH19, and PCDHGB5), showing considerably low RPKM values compared with the control embryos. Additionally, genes associated with eye development, such as RBP1, ALDH1A3, and VAX2, exhibited remarkably low RPKM values after gpx1 knockout compared with the control embryos.