Glucose-6-Phosphate Dehydrogenase, Redox Homeostasis and Embryogenesis

Normal embryogenesis requires complex regulation and precision, which depends on multiple mechanistic details. Defective embryogenesis can occur by various mechanisms. Maintaining redox homeostasis is of importance during embryogenesis. NADPH, as produced from the action of glucose-6-phosphate dehydrogenase (G6PD), has an important role in redox homeostasis, serving as a cofactor for glutathione reductase in the recycling of glutathione from oxidized glutathione and for NADPH oxidases and nitric oxide synthases in the generation of reactive oxygen (ROS) and nitrogen species (RNS). Oxidative stress differentially influences cell fate and embryogenesis. While low levels of stress (eustress) by ROS and RNS promote cell growth and differentiation, supra-physiological concentrations of ROS and RNS can lead to cell demise and embryonic lethality. G6PD-deficient cells and organisms have been used as models in embryogenesis for determining the role of redox signaling in regulating cell proliferation, differentiation and migration. Embryogenesis is also modulated by anti-oxidant enzymes, transcription factors, microRNAs, growth factors and signaling pathways, which are dependent on redox regulation. Crosstalk among transcription factors, microRNAs and redox signaling is essential for embryogenesis.


Introduction
The quality of an embryo and its growth warrant the existence and perpetuation of a species. Signaling events regulating cell proliferation, migration and differentiation with temporal and spatial precision are critical for healthy embryos. Defective embryos are subjected to destruction by various mechanisms [1]. Reduction-oxidation (Redox) status modulated by an imbalance of reactive species and anti-oxidants sensitizes signaling pathways, leading to altered cellular fates and embryonic defects [2]. Classic studies of embryogenesis have focused on the deleterious effects of oxidants on lipids, proteins and nucleic acids. Oxidants can serve as signaling molecules by initiating and promoting cell proliferation during embryonic development [3].
Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in the pentose phosphate pathway (PPP). G6PD is ubiquitously expressed in living organisms. The biochemical functions of G6PD include the oxidation of glucose-6-phosphate to

The Role of Redox in Humans and Other Species
Different animal models have been used in different redox studies because redox systems in all species are somewhat similar. In mammalian cells, the main source of intracellular ROS production are the NOX family (NOX 1-5, DUOX 1/2) [24] and the mitochondrial electron transport chain (ETC complexes I-IV, electron transporters ubiquinone and cytochrome c) [25][26][27]. Sequence comparisons and phylogenetic analysis indicate that C. elegans possesses twenty proteins closely related in sequence and length to TRX in S. cerevisiae, H. sapiens, and D. melanogaster [28]. Human mitochondrial TRXN2 (an ortholog with C. elegans TRX-2) is oxidized by mitochondrial-dependent superoxide, leading to the activation of the apoptosis signal-regulating kinase 1 (ASK-1) signaling pathway [29]. In humans, there are two thioredoxin genes, TXN and TXN2. In S. cerevisiae, thioredoxins are encoded by TRX1, TRX2 and TRX3 [30]. In addition, a family of anti-oxidant proteins, peroxiredoxins (Prx), are conserved in all types of organisms, including bacteria, plants, nematodes, and mammals. C. elegans peroxiredoxins-2 (CePrx2), a homolog of human PRDX2, is essential for normal growth and egg production [31].
In bacteria, fungi, worms and mammals, the disulfide reducing enzymes, GSH reductase and TrxR, are conserved. However, D. melanogaster does not have GSH reductase. It is replaced by TrxR, which results in the maintenance of a high GSH concentration [32]. Glutaredoxins (GLRX) are redox enzymes that use glutathione as a cofactor for reducing protein disulfides. The mammalian GLRX3/PICOT (ortholog of C. elegans GLRX-3) is involved in immune cell activation, development of cardiac hypertrophy, embryonic development and post-embryonic growth. GLRX3 also functions as an iron-sulfur protein [28]. Another redox compound, protein disulfide isomerase (PDI), which is found in the endoplasmic reticulum (ER), catalyzes the formation (oxidation), breakage (reduction) and rearrangement (isomerization) of disulfide bonds between cysteine residues within proteins. PDI-1, PDI-2 and PDI-3 are important for embryogenesis, viability and formation of the extracellular matrix in C. elegans [33].

G6PD Studies in Embryonic Stem Cells and Animal Models
G6PD has a pro-survival role in cells and organisms [5]. How an enzyme involved in carbohydrate metabolism and redox homeostasis regulates cell survival and reproduction is largely unknown due to the biological complexity ( Figure 1).

Embryonic Stem Cells
Upon targeted inactivation of G6PD by homologous recombination, male mice embryonic stem (ES) cells are viable; however, they are very sensitive to oxidative stress [34]. Despite normal proliferation in vitro, G6PD-depleted ES cells are prone to apoptosis and mitochondrial dysfunction when challenged with oxidants. This can be ascribed to intracellular GSH levels [35]. G6PD-deficient ES cells can differentiate into many cell types, while G6PD-deficient erythroid cells undergo apoptosis upon hemoglobin switching [36]. This is caused by elevated oxidative stress due to enhanced release of oxygen from hemoglobin. Despite delaying apoptosis by the reducing agent, NAC, or a pan-caspase inhibitor zVADfmk, complementation of the G6PD gene into G6PD-deficient ES cells prevents cellular demise [36].
Cre-lox recombination-derived G6PD-deficient mouse ES cells are sensitive to oxidative stress [37]. Diamide, a potent thiol scavenger, depletes NADPH and GSH by oxidation and decreases survival in G6PD-deficient ES cells. While wild type ES cells rapidly restore the NADPH/NADP + ratio and PPP activity upon diamide challenge, G6PD-deficient ES cells fail to do so. This indicates that the activation of G6PD is the major NADPH source in response to oxidative stress.

Mouse
The importance of G6PD in embryogenesis has been shown in mouse studies [38]. G6PD(-) ES cells are used to inject into mouse blastocysts to produce chimeric mice. The first generation (F1) G6PD(+/-) heterozygotes obtained from the cross between normal females and chimeric males are healthy and fertile. Only normal G6PD males can survive when F1 G6PD(+/-) females are bred with normal males. Hemizygous G6PD(-) male embryos fail to grow at E.7.5, a critical stage for the development of the blood circulation. These embryos eventually die at E10.5. Likewise, heterozygous G6PD(+/-) females display tissue abnormalities and increased apoptotic cells. At later stages, necrotic cells appear and the embryos die between E11.5 and E12.5. Both G6PD(-) and F2 G6PD(+/-) embryos show apoptotic cells in the allantois, indicating a disturbance of placenta differentiation. The lethality of G6PD-deficient mouse embryos is ascribed to oxidative damage after the establishment of the blood circulation, thereby affecting placental function. Hence, G6PD plays a protective role against oxidative stress in the placenta to support the growth of trophoblast during embryogenesis. A comparative study shows that female mouse embryos contain less H 2 O 2 and survive better than male embryos under heat stress [39]. The slow-growing female embryos display a higher level of G6PD transcripts. Upon G6PD inhibition, the differences caused by ROS between male and female embryos are absent, suggesting a protective role of G6PD in differential sensitivity of heat-induced oxidative stress during embryogenesis. of GSH. This mechanism prevents embryonic cells in animal models from undergoing apoptosis, mitochondria dysfunction and embryonic lethality in animal models. Panel B: G6PD can act as a pro-oxidant in low oxidative stress conditions (eustress) by providing NADPH for NOX/NOS in producing ROS or RNS, which are required for ESCs proliferation, differentiation and migration.

Zebrafish
A transparent embryo and ex utero development make zebrafish an ideal model organism for studying embryogenesis. A G6PD morpholino knockdown zebrafish has been established due to the lack of significant hemolysis in G6PD-deficient mice upon oxidant challenge [40]. G6PD morpholino reduces G6PD activity in zebrafish leading to rapid hemolysis and pericardial edema. Titration of G6PD morpholino in zebrafish embryos does not cause edema and hemolysis until challenged with pro-oxidant compounds, including menthol, 1-naphthol and primaquine [40]. In addition to the edema and reduced hemoglobin in G6PD morphants, 1-naphthol increases ROS generation and erythrocyte apoptosis. Zebrafish embryonic defects caused by G6PD deficiency, including the de-creased epiboly rate and elevated cell shedding at the embryo surface, are mediated by the epithelial-mesenchymal transition (EMT) pathway [41]. E-cadherin, encoded by CDH-1, regulates cell movement and tissue formation during early embryogenesis in zebrafish [42]. Reduction of E-cadherin decreases cell adhesion and the epiboly rate. Complementation of human G6PD or CDH-1 cRNA reduces embryonic defects and the expression of epithelial surface markers, E-cadherin and β-catenin, in G6PD-deficient zebrafish embryos [41]. G6PD deficiency in zebrafish impairs redox signaling mediated by NADPH oxidase-4 (NOX4) and down-regulates Smad3 and miR-200b, which are involved in embryonic stem cell differentiation and stability [43,44]. These findings indicate that G6PD plays a cytoregulatory role in appropriating redox homeostasis for modulating embryogenesis.

Nematode
In order to decipher the common mechanism underlying early embryogenesis, a simple invertebrate animal model may be suitable for large-scale analyses using cell biology, molecular biology and genetic approaches. A pilot research program led by Sydney Brenner sought an organism with the potential to identify each gene involved in development and to trace the lineage of every cell. Caenorhabditis elegans, a free-living soil nematode, has been chosen because of several advantages [45]. C. elegans has become a favorite model of embryologists because its embryo and cuticle are transparent, thereby benefiting microscopic examination. C. elegans, approximately 1 mm in size, has a short duration of embryogenesis (16 h) and lifecycle (3 days) as well as a life span (3-4 weeks), which is ideal for investigation of the gene regulatory network in embryogenesis, development and aging.
The high degree of conservation of G6PD between the nematode and vertebrate counterparts indicates that G6PD in C. elegans is a functional homologue of vertebrate G6PD. A G6PD-deficient C. elegans established by RNA interference knockdown displays enhanced apoptotic germ cells and less embryo production, indicating impairment of oogenesis [46]. Similar to mouse and zebrafish models, G6PD-deficient C. elegans shows a dramatic reduction of hatched embryos. Such embryonic defects are possibly due to increased ROS and DNA oxidative damage, leading to altered MAPK signaling [46].
Further characterization of G6PD-deficient C. elegans embryos reveals that their membrane function is impaired. These membrane abnormalities in C. elegans embryos, including enhanced permeability to fluorescent dyes and loss of barrier structural integrity, as well as abnormal polarity and cytokinesis, are associated with altered lipid metabolism [47]. A significant increase of lysoglycerophospholipids is associated with the activation of calcium-independent phospholipase A2 (iPLA), possibly mediated by the imbalanced redox status, including decreased NADP levels and increased lipid peroxidation. Suppression of multiple iPLA alleviates defective permeability and polarity.
Transient treatment of tert-butyl hydroperoxide (tBHP), an inducer of lipid peroxidation decreases brood size and increases germ cell apoptosis [48]. tBHP stimulates malondialdehyde (MDA) generation and up-regulates iPLA activity, thereby interfering with oogenesis and embryogenesis. These findings indicate that G6PD deficiency-derived lipid peroxidation enhances germ cell demise, leading to embryonic lethality, and demonstrates that G6PD is required for maintaining membrane stability and lipid homeostasis through redox balance during early embryonic stages.

The Redox Role of G6PD in Cellular Migration and Differentiation during Embryogenesis
Oxidative stress differentially influences cell fate and embryogenesis. Physiological levels (eustress) of ROS generated during embryogenesis promote proliferation and differentiation of cells and are required for normal development in the blastocyst stage and neuronal differentiation of embryogenesis [49]. ROS is involved in crucial processes, such as embryonic blood stem cell development [50,51] and differentiation of embryonic cardiomyocytes [52][53][54]. ROS can affect stem cell fate and generate stem cells, as seen with the differentiation of mesenchymal stem cells (MSC) to adipocytes caused by the enhancement of intracellular ROS through NOX4-mediated H 2 O 2 signaling [55]. Increased ROS beyond physiological levels (oxidative stress) causes apoptosis, whereas even higher ROS levels lead to necrosis [56]. This suggests that redox-dependent signaling plays a critical role in cell migration and cell differentiation during embryogenesis (Figure 1).

Involvement of G6PD in Cell Migration
Cell migration occurs during embryogenesis. In gastrulation, groups of cells inside blastocysts migrate collectively to form the three layers of the embryo (ectoderm, mesoderm and endoderm) that eventually migrate to target locations, where they differentiate and form various tissues and organs [57]. The collective cell migration requires coordination and teamwork between migrating cells, which interact with the extracellular matrix of other tissues and respond to chemotactic signals generated in the surrounding environment [58].
One of the signals that influence cell migration during embryogenesis is redox signaling, which can alter cell fate, leading to structural and functional changes in the developmental processes. Disruption of redox signaling during organogenesis induces adverse outcomes, such as defective cell migration, premature cell differentiation, apoptosis, alterations in cell proliferation and cellular polarity [2].
Redox signaling influences cell migration during embryogenesis through different mechanisms in different animals. Up-regulation of ROS induced by ethanol decreases hemangioblast migration from the posterior primitive streak to the area of opaca in the early chick embryo via the FGF/VEGF/PDGF signaling pathway [59]. In zebrafish, glutaredoxin 2, the vertebrate oxidoreductase protein modulating the intracellular thiol pool, is required for heart development. Knockdown of glutaredoxin 2 disturbs migration of the cardiac neural crest cells, leading to apoptosis [60]. Upon knockdown of superoxide dismutase 1, elevated ROS impairs embryonic primordial germ cell migration and adhesion in cellular blastoderm embryos of Drosophila melanogaster [61].

Contribution of G6PD to Cell Differentiation
Cell differentiation is closely associated with cell proliferation in a developing embryo. Embryonic differentiation occurs after zygote cleavage. The pluripotent ES cells differentiate into many cell types followed by organizing into tissues and organs [62,63]. Cell differentiation in the developmental process is influenced by redox signaling. ROS generated by osteoclasts is important for differentiation and the resorption of bone tissue [64]. Undifferentiated pre-adipocytes have a high level of GSH, whereas the GSH level drops and GSSG increases upon differentiation [65].
NOX4-dependent ROS production regulates cardiac differentiation through the p38 MAPK signaling pathway [66]. Increased ROS by NOX4 is involved in adipocyte differentiation via CREB (cAMP response element-binding protein) in mesenchymal stem cells [55]. Enhanced ROS production occurs during carbon monoxide-induced neuronal differentiation in SH-SY5Y cells. This event stimulates the PPP to increase NADPH production and affect cell differentiation [49].

Embryogenesis Regulated by Other Redox-Related Genes, Transcription Factors, MicroRNAs, Growth Factors and Signaling Pathways
Embryogenesis is a precisely controlled biological process for reprogramming the metabolic state of the cell and driving a single cell to a mature organism [67,68]. Embryogenesis is closely associated with activation and/or repression of genes within a developmental timeline [69,70]. This process is tightly and precisely regulated by various factors, such as transcription factors, microRNAs, growth factors and signaling pathways [71][72][73][74][75] (Table 1) (Figure 2). Redox reactions are essential for providing energy production and for regulating oxidative stress. Sufficient energy production and an appropriate redox environment are required for establishing a proper milieu for the embryo. Metabolically generated oxidants and associated anti-oxidant defenses participate in the initiation of certain outcomes during embryogenesis [76].    Table 1.

Anti-Oxidant Enzymes
Several anti-oxidant enzymes are involved in embryogenesis, such as superoxide dismutase (SOD), GPx, Prx and catalase (CAT). These anti-oxidant enzymes catalyze the decomposition of hydrogen peroxide into water and oxygen [28]. SOD controls ROS levels by converting superoxide to hydrogen peroxide, which can be detoxified to water by catalase and glutathione peroxidase [106]. Protein and mRNA of SOD are expressed during bovine embryogenesis [107]. The good quality of oocytes and embryos exhibits high SOD activity [108]. SOD also prevents the motility loss in mouse sperm and protects embryos from oxidative stress [109]. Newborn mice with sod-2 -/deficiency show neonatal lethality after 4-5 days. Lacking SOD activity can be fatal in newborns [73]. These findings reveal the importance of ROS levels being regulated by an antioxidative enzyme in embryogenesis.
GPx reduces H 2 O 2 to H 2 O using GSH as the electron donor. One of the GPx isozymes, GPx4/PHGPx (phospholipid hydroperoxide glutathione peroxidase), reduces lipid hydroperoxide in cell membranes and is involved in gastrulation. GPx4 expression is upregulated by G-rich RNA Sequence Binding Factor 1 (Grsf1) through a post-transcription mechanism. Grsf1 recruits GPX4 mRNA to translationally activate polysome fractions by targeting the 5 untranslated region (UTR) of GPx4 mRNAs. Overexpression of GPX4 mRNA rescues Grsf1 deficiency-induced developmental retardation of embryos. This posttranscriptional regulation links the anti-oxidant enzyme, GPx4, to embryogenesis [102]. Homozygous GPx4-deficient mice die in utero at midgestation between 7.5 and 8.5 days post coitum because of retardation in brain development and abnormal heart formation [13,80]. However, the heterozygotes GPx4-deficient mice are viable, fertile and appear normal, in spite of having decreased levels of cytosolic, mitochondrial and nuclear GPx4 mRNA and proteins. These heterozygote GPx4-deficient mice are more susceptible to various oxidative stressors [13]. Another GPX isozyme, GPx3, is required for posterior embryogenesis in Xenopus laevis. GPx3 knockdown enhances cell death and decreased cell proliferation in the tail region through the Wnt/Notch/FGF signaling pathway [110].
Within the embryo, the nervous system is not completely developed [111]. Redox signaling contributes to the development of the nervous system, such as axonal growth and neuronal differentiation [112]. Redox imbalance can negatively affect embryonic neural development. Silencing GPx4 expression by RNA interference leads to a hindbrain developmental defect in mice (rhombomeres 5 and 6 impairment) [80]. SOD2 deficiency results in increased ROS levels, abnormal brain morphology and axonal aberration in Drosophila [79]. This suggests that embryonic development of the nervous system is redox sensitive.
Prx removes ROS through conserved reactive cysteines residues by which H 2 O 2 is reduced. The mammalian Prxs affect gamete maturation, fertilization and embryogenesis. The deficiency of Prx does not have lethal consequences for embryogenesis. Among the six members of Prxs, Prx2, Prx3 and Prx5 influence embryogenesis [81]. Prx2 stimulates the development of the blastocyst by reducing intracellular ROS and increasing embryonic mitochondrial activity [82]. Prx3 is one of the candidate genes that predict embryo quality. Lower expression of Prx3 is associated with high-quality embryos [83]. Drosophila peroxiredoxin 5 (dPrx5) is essential in the early stages of embryogenesis [84]. C. elegans with Prx-2 deficiency have a reduced brood size and retarded development. The CePrx2 only affects two types of pharyngeal neurons, which are single pharyngeal interneuron 14 and the sensory interneuron 12 [31].
Catalase is found in all living organisms. One catalase molecule can reduce millions of H 2 O 2 molecules to yield H 2 O and O 2 . The peroxisomal CTL-2-deficient C. elegans has a lower egg-laying capacity and shorter life span [113]. Embryonic catalase protects the embryo from excessive ROS production. The lack of catalase promotes structural embryopathies in C57BL/6 mice [114][115][116][117].

Transcription Factors
As key regulators of various cellular processes, transcription factors activate downstream target genes by enhancing transcription activity [118]. Transcription factors can sense cellular redox status, including NF-E2-related factor 2 (Nrf2), Nuclear factor kappalight-chain-enhancer of activated B cells (NF-κB), Activator Protein-1 (AP-1) and hypoxiainducible factor (HIF), thereby contributing to the regulation of cell proliferation, differentiation and apoptosis in embryos [76].
Nrf2 regulates the adaptive response to oxidative stress [119]. The evolution of Nrf2 is correlated with increasing atmospheric oxygen [120]. Nrf2 binds with genes containing the anti-oxidant response element (ARE) [121]. Nrf2 also serves as a master regulator for selfrenewal, pluripotency and differentiation in ES cells [122]. In mesodermal development, multipotent stromal cells (MSCs) differentiate to osteoblasts, chondrocytes, myocytes and adipocytes [123]. Overexpression of Nrf2 in MSCs enhances oxidative stress resistance, cell survival and osteoblastic differentiation [87,88]. In early embryogenesis in mice, the Nrf2-associated cell cycle transition from the G2 to the M phase is inhibited by brusatol (a quassinoid isolated from Bruceajavanica), and this inhibition is dependent on the cyclin B-CDK1 (cyclin-dependent kinase I) complex [89]. This indicates that a dual role exists for Nrf2 in maintaining redox homeostasis and modulating embryogenesis.

Redox-Related Post-Transcriptional Controls
Post-transcriptional regulation plays a crucial role in redox-dependent developmental processes. Myogenesis is an important biological process for forming skeletal muscular tissue during embryogenesis [127]. Loss of function of two transcription factors, Paired-like homeodomain transcription factor 2 gene (Pitx2) and Pitx3, leads to abnormal accumulation of ROS in mouse myogenesis [100]. The Pitx-MicroRNA (mir-15b, mir-23b, mir-106b and mir-503) pathway regulates myoblast proliferation, demonstrating that the crosstalk of transcription factors, microRNAs and redox signaling is essential for embryogenesis [101].
The basic helix loop helix (bHLH) and Per-ARNT-SIM (PAS) transcription factor superfamily proteins of HIF are essential in developmental programs, such as trophoblast proliferation and differentiation, morphogenesis of the developing heart, chondrogenesis and myocardium development [96][97][98][99]. They activate various downstream target genes via the hypoxia response element in the enhancer region. Among three HIF-α subunits, HIF-1α is ubiquitously localized in developing tissues and is regulated by the oxygen gradient [128]. HIF-1α is regulated by post-transcriptional control. The 3 or 5 UTR of HIF-1α mRNA contains AU-rich elements attracting the RNA binding protein, such as Human antigen R (HuR) or polypyrimidine tract-binding protein (PBP), for stabilizing HIF-1α mRNA and stimulating HIF-1α protein synthesis [129,130]. Similarly, the 3 UTR of Trx mRNA is protected from degradation by the cold-inducible RNA-binding protein (CIRP) hnRNPA18, which enhances Trx expression [131]. Such post-transcriptional regulation is found in the mRNA of redox-embryogenesis related enzymes, including Cox-2, GPx1 and MnSOD (manganese-dependent superoxide dismutase or mitochondrial SOD2) [76].

Growth Factors and Redox Modulation
Growth factors contribute to cell proliferation and differentiation in embryogenesis [132]. The signaling of the epidermal growth factor receptor (EGFR) shows a redoxregulated linkage to tyrosine kinase signaling, which is redox-sensitive [133]. Fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) are inhibited in hyperoxia, leading to bronchopulmonary dysplasia in humans, demonstrating a linkage between ROS, growth factors and lung development [75]. H 2 O 2 -induced pulmonary hypertension in rats, which is modulated by EGFR signaling through redox regulation [134].
Vasculogenesis is an important event during early mammalian embryogenesis. In order to supply nutrients for embryos, blood vessels are stimulated for growing, branching and invading developing tissues and organs [135]. Crosstalk between growth factors and redox-related transcription factors promotes vascular development. HIF-1 responds to systemic oxygen changes, activates vascular endothelial growth factor (VEGF) transcription and modulates vascular development for adaption to hypoxia. HIF-1 is protective during embryogenesis, as demonstrated by preventing the lethality of mice at the embryonic 10.5 stage (E10.5) [56,136].

Redox Status and Signaling Pathways
The redox status plays a critical role in regulating differentiation, proliferation, apoptosis and cell division asymmetry during embryogenesis [2]. Therefore, redox signaling is required for building a healthy embryo at the correct time point and place. The evolutionally conserved regulation of the O 2 concentration controls cell metabolic processes by producing, sensing and responding to its metabolites (ROS). This regulation contributes to redox cellular signaling. This network of redox cellular signaling not only involves cell metabolism but also embryogenesis [137]. ES cells undergo cell cycle arrest of the G2/M cell cycle phase transiently by increasing ROS levels, which indicates the oxidative stress resistance of the ESCs [138]. ES cells at an O 2 concentration of 2-5% maintain their clonal regression and genetic integrity [139,140]. Lower O 2 concentrations or oxidative stress induced by H 2 O 2 leads to ES cell death by activating apoptotic signal transduction [138,140], indicating the importance of maintaining a physiological O 2 concentration for regulating redox signaling for directing ES cells into the correct timeline of the cell cycle.
The Wingless and int-1 (Wnt) signaling pathway regulates embryonic cell differentiation, proliferation, cell fate determination and apoptosis, especially at the gastrulation stage [141,142]. Wnt signaling is activated by sensing the ROS level. Nucleoredoxin (Nrx) acts as a key regulator for Wnt signaling regulation by targeting dishevelled (Dvl) activity directly [105]. Hydrogen peroxide oxidizes Nrx, leading to Dvl release from the Nrx/Dvl complex, and promotes β-catenin accumulation to activate Wnt signaling [104]. Differential levels of Nrx regulate Wnt signaling during Xenopus gastrulation [103].

Conclusions
Maintaining redox homeostasis is critical for the normal development of an embryo. NADPH produced from the action of G6PD is important in maintaining redox homeostasis by serving as a cofactor for NADPH-dependent redox enzymes, including GSH reductase, TrxR, aldo-keto reductase, as well as NOX and NOS. GSH and TRX function as anti-oxidants that contribute to the maintenance of redox homeostasis, while ROS and RNS at physiologic concentrations (eustress) contribute to cellular growth and differentiation. G6PD is involved in oogenesis, embryogenesis and post-embryonic development. This is substantiated from studies in which G6PD is inactive or knocked-down. G6PD-deficient cells and organisms have been used as models for determining the role of redox signaling in regulating cell proliferation, differentiation and migration during embryogenesis. Embryogenesis is also modulated by anti-oxidant enzymes, transcription factors, microRNAs, growth factors and signaling pathways, which are dependent on redox regulation. Novel technologies, including multi-omics sequencing [143] and single-cell sequencing [144], are promising for comprehensively uncovering the molecular actions of how redox homeostasis regulates embryogenesis. Recent development of genetic engineering tools, such as CRISPR knock-in, can further establish new G6PD models in stem cells and model organisms [145][146][147]. It is anticipated that other redox-sensitive pathways governing embryogenesis will be reported following the development of novel and advanced technologies.