Abstract

Calcium signaling is essential for embryonic development but the signals upstream of calcium are only partially understood. Here, we investigate the role of the intracellular glutathione redox potential in calcium signaling using the Chac1 protein of zebrafish. A member of the γ-glutamylcyclotransferase family of enzymes, the zebrafish Chac1 is a glutathione-degrading enzyme that acts only on reduced glutathione. The zebrafish chac1 expression was seen early in development, and in the latter stages, in the developing muscles, brain and heart. The chac1 knockdown was embryonic lethal, and the developmental defects were seen primarily in the myotome, brain and heart where chac1 was maximally expressed. The phenotypes could be rescued by the WT Chac1 but not by the catalytically inactive Chac1 that was incapable of degrading glutathione. The ability of chac1 to alter the intracellular glutathione redox potential in the live animals was examined using Grx1-roGFP2. The chac1 morphants lacked the increased degree of cellular oxidation seen in the WT zebrafish. As calcium is also known to be critical for the developing myotomes, brain and heart, we further investigated if the chac1 knockdown phenotypes were a consequence of the lack of calcium signals. We observed using GCaMP6s, that calcium transients normally seen in the developing embryos were strongly attenuated in these knockdowns. The study thus identifies Chac1 and the consequent change in intracellular glutathione redox potential as important upstream activators of calcium signaling during development.

Introduction

Calcium signaling is vital for both early and late stages of embryonic development [1]. Early in development at the blastomere stage and in the later segmentation phases, calcium signaling is thought to be largely intracellular [2], while in the intervening gastrulation phase, it is largely intercellular signaling [3]. At later stages, the role of calcium signaling has been clearly demonstrated for the developing brain [4,5], heart [6] and myotomes [7]. The activators upstream of calcium mobilization are, however, not very well known, especially during development. NAADP, cADPR and IP3 are known activators of calcium signaling and can activate the Ryanodine receptor and two-pore channels, both of which are important for proper development [811]. Although calcium channels are also known to be regulated by the redox potential [12,13], the possibility that changes in the redox potential might play a role in altering calcium fluxes during development has never been adequately investigated owing to the difficulty in creating well-determined changes in redox in developing embryos.

The recent discovery of two new glutathione degrading enzymes, ChaC1 [14] and ChaC2 [15], members of the γ-glutamylcyclotransferase family that are cytoplasmically located, and that act only on reduced glutathione reveals a mechanism for changing the redox milieu. These enzymes of glutathione degradation, owing to their selectivity towards reduced glutathione, when induced can provide cells with a more controlled way of regulating redox [16]. Studies with heterologously expressed mammalian ChaC1 in yeast cells showed that ChaC1 expression led to an increase in the degree of oxidation (OxD) of the cells and the activation of both TRP-like and L-type calcium channels by specific glutathionylation [17,18]. Here, we have investigated the link between calcium and redox during development using the Chac1 protein of zebrafish. This protein was also capable of degrading glutathione and altering the intracellular glutathione redox potential of cells by generating a more oxidizing environment. The zebrafish chac1 was found to be expressed early in development, but the subsequent expression was largely restricted to the developing brain, heart and the myotome. The developmental defects of the chac1 knockdown were seen primarily in the development of the brain, heart and the myotome. Since calcium signaling has been previously shown to play important roles in the development of these organs, we analyzed both the intracellular glutathione redox potential and the calcium transients in the developing zebrafish. An absence of the oxidizing environment and an almost complete lack of calcium transients were observed with the chac1 knockdown. Our results suggest that the Chac1-mediated specific degradation of reduced glutathione and the consequent rise in the intracellular glutathione redox potential is required for the calcium signaling that is essential for the developing zebrafish.

Materials and methods

Chemicals and reagents

All chemicals used in this study were either analytical or molecular biology grades and were obtained from commercial sources. Media components were purchased from Sigma–Aldrich and Difco, U.S.A. Dithiothreitol (DTT) and Diamide purchased from Sigma–Aldrich, U.S.A. Oligonucleotides were purchased from IDT, U.S.A. H2O2 purchased from Merck, Germany. Oxidized and reduced glutathione purchased from Sigma–Aldrich, U.S.A.. Restriction enzymes and DNA polymerase enzyme were purchased from New England Biolabs (NEB), U.S.A. Gel extraction kits and plasmid miniprep columns purchased from Promega, U.S.A. In vitro SP6 Transcription Kit, Thermo Fisher, PVDF Blotting membrane (P0.45) purchased from GE Healthcare Life science, U.S.A. Western HRP substrate was obtained from Millipore, India.

Zebrafish maintenance

Zebrafish experiments were carried out in the laboratory of Rajesh Ramachandran at IISER Mohali. Zebrafish were maintained at 28°C on a 14 h light and 10 h dark cycle in a closed circulating fish water system (Aquatic Habitats) and adult zebrafish were fed a complete granular diet (FRIPPAK; INVE, Thailand) or live Artemia cysts (Sanders) twice a day. By natural mating of wild-type (AB strain), we obtained embryos, which were further maintained in a petri dish containing fish water until they reached the proper stages for observation. Developmental stages were ascertained according to the hours post fertilization (hpf) and morphological characterization as described [19].

RNA preparation and real-time quantitative polymerase chain reaction (qRT-PCR)

Total RNA was isolated from wild-type whole embryos at different stages of development using TRIzol (Sigma; Cat. No. T9424) and was quantified in a Nanodrop spectrophotometer (Jenway). Following the manufacturer's instructions (Promega, Cat. No. A5000), up to 5 µg of total RNA per sample was used to synthesize cDNA using Reverse Transcriptase. Pre-amplification of chac1 was done using RED Taq PCR master mix (Sigma, Cat. No. R2523) with 0.5 µM gene-specific primers and 0.5 µl cDNA. The resulting cDNA (0.5 µl) was amplified using primers specific for chac1 (forward and reverse primer, 5′-CATCGTGGAGATGATGAAATGCCCG-3′ and 5′-CGATGTAAACCAGAGCCTGGACTGG-3′, respectively) and β-actin2 (forward and reverse primer, 5′-GCAGAAGGAGATCACATCCCTGGC-3′ and 5′-CATTGCCGTCACCTTCACCGTTC-3′, respectively). The β-actin was used as a positive control to confirm consistent quality and quantity of cDNA. PCR reactions were done using Vent DNA Polymerase (NEB) under the following conditions: denaturation at 95°C for 5 min, 30 cycles of 95°C for 30 s, annealing at 62°C for 30 s and extension at 72°C for the 30 s. The qPCR reactions (20 µl) were run in triplicates with a Syber green qPCR supermix (Invitrogen; Cat. No.11784200) and primers at 0.5 µM. The PCR cycle was performed according to the manufacturer's instructions (Roche thermo cycling). The conditions followed were 5 min at 95°C, 40 cycles of 95°C for 30 s, annealing temperature for each primer for 30 s and 72°C for 30 s and a melting curve program from 55 to 95°C with 0.5°C change in 10 s interval was applied. All reactions were run in triplicates, and GraphPad Prism5 was used for the analysis of data and statistics were carried out on raw ΔCt values [20]. qRT-PCR data were represented as the mean ± s.e.m. (n = 3), P < 0.05, Student's t-test.

Morpholino oligonucleotides and mRNAs microinjections

Lissamine-tagged antisense chac1 morpholino oligonucleotides (MOs) were purchased from Gene Tools, LLC. The chac1MO (chac1MO was designed to bind to the 5′-UTR including the start codon, AUG) 5′-TGTGCCTTTTGCTATTGCGACCTC-3′ and chac1MO2 5′-TGTGCGCTTTGATGTAAAAACAGCT-3′ (chac1MO2 was designed to bind to the 5′-UTR sequences near to the start codon, AUG but not overlapping with chac1MO) complementary to the 5′ sequence of chac1 were used for blocking translation. MOs were resuspended in nuclease-free water (Sigma) at 1 mM stock concentration. Both chac1 and control MO were injected at a 100–500 µM final concentration at one- to two-cell stage embryos using a Femtojet4X microinjector (Eppendorf), and standard MO 5′-CCTCTTACCTCAGTTACAATTTATA-3′ was used as a control MO as has been previously described [21]. Each MO injection was repeated at least three times and the doses were optimized for better phenotypes. Eventually, we chose a dose that enabled viability that allowed us to investigate the phenotypes. For mRNA microinjection, all mRNAs were transcribed in vitro transcription using the mMessage mMachine in vitro SP6 Transcription Kit (Thermo Fisher Cat No. AM 1340) from their corresponding ORF sequences in the pCS2 vector. Purified by lithium chloride precipitation and dissolved in nuclease-free water and quantified by Nanodrop. For the rescue experiments, we co-injected chac1MO and chac1 mRNA where the sequence of the chac1 mRNA or catalytically inactive mutant chac1 mRNA (E95Q) did not contain any overlap with the chac1MOs. We have used a range of mRNA concentration (10, 50, 100 and 150 ng) for optimization. At higher concentrations, we observed deleterious effects and thus 100 ng was used in the rescue experiments. After injection of MOs, embryos were maintained in fish water until they reached the stages for observation. The number of embryos that were used for knockdown and rescue experiments are mentioned in Figure 3F.

Confocal and stereo fluorescence microscopy

Fluorescence and DIC images were acquired with LSM 700 (Zeiss) Confocal Laser Scanning Microscope (Achromate plan 10×/NA 0.8 objective lenses). Both dechorionated and with chorion zebrafish embryos were selected at the various developmental stages (temperature ≈ 25°C) and were placed in a glass-bottom 35 mm petri dish (Eppendorf) and mounted in 3% methyl cellulose (Sigma–Aldrich) and acquired the images of different stages of development. The MOs-injected or wild-type embryos were observed for morphological changes under a Zeiss AxioScope A1 Stereomicroscope (Fluorescence and bright field Microscope) at various developmental stages [19]. Furthermore, we evaluated the developmental defect in various organs in the morphants and compared with wild-type embryos. All images were processed using Fiji (ImageJ) (http://fiji.sc).

Optical cutting temperature embedding, sectioning of embryos and staining

Wild-type and chac1MO embryos of 72 hpf were fixed in 4% of paraformaldehyde (PFA) for overnight incubation at 4°C. For embedding, embryos were dipped in optical cutting temperature at required orientation and were kept overnight at −80°C. 10 µm sections were cut using the Leica RM2155 microtome and counterstained with Harris hematoxylin and eosin (H & E) for observing muscle morphology in a routine manner [22]. Images were taken using an EVOS (Life technologies) microscope.

Cloning of zebrafish Chac1 protein-encoding gene in the yeast vector

Zebrafish chac1 was PCR amplified from cDNA and cloned into the yeast expression vector p416 TEF. Site-directed mutagenesis was performed using the Splice Overlap Extension PCR (SOE PCR) method. The mutant created at position E95Q in zebrafish chac1 was sequenced to confirm the presence of the desired nucleotide change. Primers used for cloning include forward primer (5′-ATCGCGGATCCACCATGAAACCTCAAGACATCGTCGC-3′) and reverse primer (5′-ATCCGCTCGAGCTATGCGGCCAATAATATGGGTC-3′) and for site-directed mutagenesis, the primers used include forward primer (5′-CTGAACGTGAGGCAGGCAGTGAGAGGT-3′) and reverse primer (5′-CACCTCTCACTGCCTGCCTCACGTTCAG-3′). The chac2 was custom synthesized from Synbio Technologies and subcloned into a yeast expression vector (p416 TEF).

Yeast transformation and spotting based growth assay

Yeast (Saccharomyces cerevisiae; strains used: BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0), and ABC1723 (met15Δecm38Δdug3Δ in BY4741) transformations were carried out using the lithium acetate method [23]. In brief, the yeast extract, peptone and dextrose medium was used for routine growth and transformation. Transformants were selected on synthetic defined (SD) minimal medium containing yeast nitrogen base, ammonium sulfate, dextrose supplemented with auxotrophic amino acids or glutathione as required. The overnight grown primary cultures were re-inoculated and grown at 30°C until OD600 nm = 0.6–0.8, harvested, washed with distilled water and resuspended in distilled water to OD600 nm = 0.2. Serial dilutions were prepared, and 10 μl of each dilution was spotted onto the SD medium containing amino acid supplements plus either 300 µM reduced glutathione (GSH) plates or 300 µM methionine as required. The plates were incubated at 30°C for 2–3 days and photographs were taken using a Bio-Rad molecular imager Gel Doc imaging system.

Bioassay for: To demonstrate the ability of Chac1 to degrade glutathione bioassay was used.

In the assay, ABC1723 was used. This strain (owing to a met15Δ) is unable to utilize inorganic sulfate and is an organic sulfur auxotroph which is able to grow on sulfur sources like methionine and glutathione. The growth of glutathione requires the degradation of glutathione. The strain also carries deletions in two of the enzymes known to be involved in this degradation (γ-glutamyl transpeptidase (GT), ECM38 and the Dug enzyme component, DUG3). Thus, growth on glutathione reflects the ability of Chac1 to degrade glutathione [14].

Whole-mount mRNA in situ hybridization

To prepare digoxigenin-labelled chac1 antisense mRNA probe (FL/DIG RNA labeling kit, Roche Diagnostics), the chac1 gene was cloned into the pCS2(+) vector and this template plasmid was then linearized by BamH1 digestion and transcribed with T3 RNA Polymerase. The in situ hybridization protocol was adapted from a previously described method [24]. On the first day, after rinsing and refixation, the embryos were prehybridized for 2 h at 56°C and then incubated in hybridization buffer (TEN solutions, 50% Formamide, 5% Dextran Sulfate, 1% RMB blocker and 500 ng–1 µg probes) overnight. On the second day, after incubation in hybridization buffer, the embryos were washed twice in Solution I (50% Formamide, 2× SSC) at 65°C and thrice in Solution II (2× SSC) at 37°C. Following this, embryos were RNaseA treated, washed twice with 2× SSC at 37°C and with 1× PBST at room temperature (RT). The embryos were then kept in blocking solution (1× Maleate, 0.05% Triton and 1% RMB blocker) for 2 h at RT and incubated overnight at RT with anti-DIG-AP antibody at 1 : 2500 dilutions in blocking solution. On the third day, after washing embryos twice with 1× Maleate and twice with Genius 3 buffer (100 mM Tris–HCl,100 mM NaCl and 50 mM MgCl2) containing 0.1% Tween, staining was done using conventional Nitroblue tetrazolium Chloride (NBT)/5-bromo-4-chloro-3-indolyl phosphate precipitation by alkaline phosphatase. For detection of chac1 expression patterns, embryos were collected at 0.25, 6, 12, 24, 48 and 72 hpf. Embryos that developed past 12 hpf were pretreated with 100 μM phenylthiourea (PTU) to prevent pigmentation before fixation in 4% PFA and manually dechorionated and fixed in 4% PFA in 1x PBS at 4°C for 7–9 h and then treated with 100% methanol. Finally, they were washed with PBS. The stained embryos were kept in 1× PBS, and images were captured by Leica Stereo Zoom Microscope (M205C, Leica Microsystems GmbH, Wetzlar, Germany) with a digital camera (Leica MC120HD).

Whole-mount immunostaining of zebrafish embryos

The whole-mount immunostaining protocol was adapted from a previously described method [25]. In brief, different developmental stages of embryos were fixed with 4% PFA at RT for 4 h and washed with 1× PBS followed by permeabilization with 1× PBS containing 0.05% Triton X-100 (1× PBST). Subsequently, the samples were blocked with 5% BSA in 1× PBST for 3 h at RT and incubated with the anti-CHAC1 antibody (human) (1:500 dilutions) for overnight at 4°C. After washing with 1x PBST, the embryos samples were incubated with Secondary antibodies Alexa Fluor 555-conjugated anti-rabbit (1:1000 dilutions) for 3 h and washed with 1× PBST. After this, the embryo samples were dried on slides in dark prior to cover-slipping with DABCO mounting medium. Images were equipped with a Nikon A1 confocal imaging system with 10× or 20× for all images. All confocal images were processed using Fiji (ImageJ) (http://fiji.sc).

Measurement of intracellular glutathione redox potential using Grx1-roGFP2 in zebrafish

For imaging of changes in the glutathione redox potential, the plasmid Grx1-roGFP2 was used. This probe has a fusion of the glutaredoxin Grx1 to the redox-sensitive GFP, Grx1-roGFP2 allowing specific real-time equilibration between the probe and the glutathione redox potential [26,27], originally obtained from Dr Tobias P. Dick was subcloned into the BamHI and XbaI site of pCS2(+) vector, linearized by KpnI digestion and capped mRNA was synthesized by in vitro transcription. Approximately, 2–3 nl of a solution containing 100 ng/μl Grx1-roGFP2 mRNA and chac1 MO (500 µM) were co-injected in one- to two-cell stage zebrafish embryos. For all the imaging, embryos were maintained in fish water. Time-lapse images were captured for 3 min [27]. We excited the Grx1-roGFP2 sequentially line by line with 405 and 488 nm and GFP emission spectra were acquired at 500–530 nm. All images were acquired at 25°C using glass-bottom dish containing 1% low-melting Agar and a TCS Leica SP8 Confocal Microscope with 10×, 0.4NA objective (Argon and UV diode LASER light source). Generally, the imaging setup was optimized for minimal LASER power to embryos during time-lapse imaging (pixel dwell time = 0.60 µs). Raw data were exported to 32 bit .tiff and were analyzed using the software Fiji (ImageJ). Mean intensity values were measured in the 405 and 488 nm channel, and the background was subtracted. The background-corrected values for the 405 and 488 nm were divided to obtain the 405/488 ratio acquired in the region of interest (ROI) by the selected area. This ratio was normalized to the mean ratio measured after reduction with DTT (R/RDTT). The spectral pseudocolor look-up table ‘fire’ (Fiji) was used to display images.

Calculations of degree of oxidation and glutathione redox potential (EGSH)

To calculate fluorescence intensity ratios, we divided the 405 nm image by the 488 nm pixel by pixel and normalized the values to the fully reduced (DTT) embryos, Rnormalized = 0.1 for comparison between different experiments. And we calculated dynamic range, which shows the maximum achievable redox changes in embryos. Dynamic range = intensity ratio of 405/488 nm of the fully oxidized (Rox) form with H2O2 100 mM divided by ratio of 405/488 nm of the fully reduced (Rred) form with 25 mM DTT. Furthermore, we calculated the OxD of Grx1-roGFP2, for that, we used the Nernst equation as described earlier in Morgan et al. [28], which gives the OxD of the Grx1-roGFP2 in an oxidized form.

The following equation was used to calculate the OxD of the Grx1-roGFP2. 
formula
Furthermore, we calculated the intracellular glutathione redox potential (EGSH) using midpoint redox potential (E0), the standard redox potential of Grx1-roGFP2 has been determined as −280 mV [29]. 
formula
where R is the gas constant (8.315 J/K), T is the absolute temperature (298.15 K), n is the number of transferred electrons and F is Faraday constant (96 485 C/mol).

Measurement of intracellular glutathione redox potential using Grx1-roGFP2 and chac1 in yeast

Saccharomyces cerevisiae (WT) cells transformed with vector and zebrafish chac1 along with Grx1-roGFP2 [28] were grown in the absence of glutathione until OD600 nm = 0.6–0.8. Exposure to 2 mM H2O2 and 10 mM DTT was taken as a control for fully oxidized and fully reduced form, respectively. The time-lapse images were taken for 3 min. We excited the Grx1-roGFP2 sequentially line by line with 405 and 488 nm and GFP emission spectra were acquired at 500–530 nm. All images were acquired at 25°C using cells mounted on a glass slide layered with a thin film of 1% agarose and a TCS Leica SP8 Confocal Microscope with 63× with immersion oil, 1.4NA objective (Argon and UV diode LASER light source). The imaging set-up was optimized for minimal LASER power to yeast cells during time-lapse imaging (pixel dwell time = 0.60 µs). This ratio was normalized to the mean ratio measured after reduction with DTT (R/RDTT). Subsequently, we calculated OxD and EGSH and represented as OxD.

Imaging cytosolic Ca2+ with GCaMP6s, processing and analysis

For imaging cytosolic Ca2+, we have used the GCaMP6s probe. The GCaMP6s is an ultrasensitive green-fluorescent protein derived protein for in vivo calcium detection [30]. The plasmid GCaMP6s was originally obtained from Dr Douglas, was subcloned into the BamHI and EcoRI site of pCS2(+) vector, linearized by NsiI digestion and capped mRNA was synthesized by in vitro transcription. Approximately, 2–3 nl of a solution containing 100 ng/µl GCaMP6s mRNA was injected into one- to two-cell stage embryos, and live imaging was performed with a confocal microscope (Zeiss LSM780) equipped with a 10× lens at various developmental stages (10, 20, 26 and 48 hpf). Calcium imaging of 10–26 hpf embryos of chac1 morphant, embryos expressing GCaMP6s reporter together with chac1 morpholino in 10–26 hpf zebrafish embryos were dechorionated manually, and mounted in 3% methyl cellulose or 1% low-melting agarose poured onto a cavity glass slide (dimension 75 × 25 mm; size: 1.4 mm; HIMEDIA) for image acquisition. The time-lapse images were acquired with a 7.5 s interval and presented as time-lapse videos (Pixel dwell time = 0.79 µs). For quantification of GCaMP6s fluorescence intensity, we placed square-shaped at EVL layer in 10 distinct ROI of zebrafish embryos (10 hpf) where GCaMP6s was expressed. Subsequently, we determined the calcium spike of EVL layer at 10 hpf embryos and later stage, calcium spike and transient at midbrain–hindbrain boundary (MHB) region zebrafish embryos at 20 hpf and we also record the calcium spike at myotome region of embryos at 26 and 48 hpf. Furthermore, we compared wild type with chac1 knockdown morphants. Similarly for 20 and 26 hpf, we placed square-shape ROI at 10 distinct areas of brain part and MHB, respectively. The square-shaped ROIs are sufficient to detect intracellular Ca2+ spike. For each ROIs, the mean fluorescence intensity was calculated and used to represent the fluorescence intensity (in arbitrary units). All images were adjusted, assembled and analyzed using Fiji (ImageJ).

Western blot analysis

Embryos were collected in Laemmli-Buffer (2 embryos per μl), sonicated (BioruptorPico, 10 cycles, 30 s on/off) or crushed with a pestle (mechanical gridding), then stored overnight at −80°C. After thawing, samples were centrifuged at 13 000 rpm for 10 min to pellet all insoluble materials. Subsequently run on SDS PAGE gel and transferred to PVDF membrane using Semi Dry-Blot (Bio-Rad). About 15 embryos were loaded per lane. Blots were blocked in PBS containing 0.1% Tween-20 (PBST) and 5% skimmed milk followed by incubation with the primary antibody diluted in PBST 1 : 3000 dilution for anti-CHAC1 (human) polyclonal antibody (Sigma–Aldrich, Cat No. AV 42623, Lot No. QC54122) and 1:2000 dilution for Anti-GAPDH (Zebrafish) (NOVUS, Cat No. NB300-221H; Lot No. 033116) for overnight and washed extensively. Furthermore, it was incubated with an anti-rabbit (Sigma–Aldrich, Cat. No. A0545; Lot. No. 022M4811) or anti-mouse (Cell Signaling Technology, Cat. No.7076S; Lot. No. 31) HRP-conjugated secondary immunoglobulin G (IgG) at RT for 2 h and washed extensively. The ECL signal was recorded using Immobilon Western Chemiluminescent HRP Substrate (MILLIPORE Cat No. WBKCS0100).

Statistical analysis

Statistical analyses were performed using GraphPad Prism5 for all the data which have numerical values. Numbers of embryos and statistical tests are indicated in the figure legends. The average value and standard error of the mean (s.e.m.) were calculated and indicated on the figures. For comparison of data, P values were determined by Student's t-test (two-tailed, unpaired) with unequal variances. P values <0.01, 0.001 and 0.0001 were considered to be significant and indicated by ‘*’, ‘**’ and ‘***’ respectively.

Results

Zebrafish has two members of the Chac family, Chac1 and Chac2, involved in glutathione degradation

The zebrafish genome reveals two Chac proteins, ChaC1 and Chac2 that are members of the γ-glutamylcyclotransferase family. The proteins were orthologous to the human CHAC1 (61% identity) and CHAC2 (72% identity) proteins, respectively. The ChaC1 and Chac2 proteins of zebrafish were both found to be glutathione-degrading enzymes, as seen through functional in vivo assays in yeast. Chac1 appeared to degrade glutathione more efficiently than Chac2, similar to what has also been observed with both the mouse and human homologs [14,15]. The catalytic residue responsible for the enzymatic activity of γ-glutamyl cyclotransferases was also conserved in the zebrafish proteins (Figure 1A), since mutation of the catalytic glutamate residue [14,15] of Chac1 to glutamine (Chac1E95Q) showed a complete loss in its activity (Figure 1B). A critical property of the Chac1 proteins is that the enzyme is specific to only reduced glutathione and not to oxidized glutathione, thus leading to an increase in the glutathione redox potential upon expression of the Chac1 protein. To confirm if the same property is observed in the zebrafish Chac1 protein, we evaluated the intracellular glutathione redox potential in cells using Grx1-roGFP2 where the zebrafish Chac1 in yeast was expressed. We observed a significant increase in the OxD of the cytoplasmic redox milieu confirming that the Chac1 protein was specific for reduced glutathione and was capable of increasing the glutathione redox potential in the cells (Figure 1C).

Zebrafish chac1 as a glutathione degrading enzyme and a regulator of redox, and its spatiotemporal expression.

Figure 1.
Zebrafish chac1 as a glutathione degrading enzyme and a regulator of redox, and its spatiotemporal expression.

(A) Sequence alignment of zebrafish (Dr), human (Hs), rat (Rn) and mouse (Mm) CHAC1 proteins. The alignment was carried out using the MUSCLE algorithm, the catalytical residue is boxed. (B)The chac genes functionally assayed for glutathione degradation with glutathione as a sole organic sulfur source. Genes were transformed into a yeast strain that is an organic sulfur auxotroph (met15Δ) and defective in enzymes for glutathione degradation thus making it dependent on Chac1 for growth on external glutathione. Growth was examined by serial dilution. Right to left: empty vector, human CHAC1, zebrafish chac1 and chac2 and chac1E95Q. (C) Zebrafish chac1 expression in yeast results in a more oxidizing environment. Wild-type yeast Saccharomyces cerevisiae cells were transformed with empty vector and chac1 were co-transformed with a plasmid containing Grx1-roGFP2 and Grx1-roGFP2 response was followed for 3 min. Exposure to 2 mM H2O2 and 10 mM dithiothreitol (DTT) was taken as a control for fully oxidized and fully reduced glutathione. The ratio of the fluorescence emission at 405 and 488 nm at fixed excitation of 510 nm is plotted against time (s). The graph shows the mean ratio of the images from three independent experiments. In each experiment, 500 cells were measured. Error bars represent s.e.m. from the mean. (D) Real-time PCR of chac1 during early embryonic development at 0.25 hpf (one-cell stage) and 6 hpf (shield stage). (E–G) Whole-mount mRNA in situ hybridization of chac1 in wild-type embryos at early stages of development with a specific probe for chac1, The chac1 is maternally provided as seen at 0.25 hpf and is ubiquitously expressed until mid-gastrulation; it has also been seen in somite boundary during segmentation. (H–R) At later stages, chac1 becomes restricted to the forebrain, midbrain (MB), midbrain–hindbrain boundary (MHB), hindbrain (HB), telencephalon (Tel), retina and the myotome. It is also found in myotome, otic vesicle (OV), lens and heart at later stages of development. Scale bar: (E–H,N,Q), 100 µm; (I,J,L,M,O,P), 50 µm; (R), 25 µm.

Figure 1.
Zebrafish chac1 as a glutathione degrading enzyme and a regulator of redox, and its spatiotemporal expression.

(A) Sequence alignment of zebrafish (Dr), human (Hs), rat (Rn) and mouse (Mm) CHAC1 proteins. The alignment was carried out using the MUSCLE algorithm, the catalytical residue is boxed. (B)The chac genes functionally assayed for glutathione degradation with glutathione as a sole organic sulfur source. Genes were transformed into a yeast strain that is an organic sulfur auxotroph (met15Δ) and defective in enzymes for glutathione degradation thus making it dependent on Chac1 for growth on external glutathione. Growth was examined by serial dilution. Right to left: empty vector, human CHAC1, zebrafish chac1 and chac2 and chac1E95Q. (C) Zebrafish chac1 expression in yeast results in a more oxidizing environment. Wild-type yeast Saccharomyces cerevisiae cells were transformed with empty vector and chac1 were co-transformed with a plasmid containing Grx1-roGFP2 and Grx1-roGFP2 response was followed for 3 min. Exposure to 2 mM H2O2 and 10 mM dithiothreitol (DTT) was taken as a control for fully oxidized and fully reduced glutathione. The ratio of the fluorescence emission at 405 and 488 nm at fixed excitation of 510 nm is plotted against time (s). The graph shows the mean ratio of the images from three independent experiments. In each experiment, 500 cells were measured. Error bars represent s.e.m. from the mean. (D) Real-time PCR of chac1 during early embryonic development at 0.25 hpf (one-cell stage) and 6 hpf (shield stage). (E–G) Whole-mount mRNA in situ hybridization of chac1 in wild-type embryos at early stages of development with a specific probe for chac1, The chac1 is maternally provided as seen at 0.25 hpf and is ubiquitously expressed until mid-gastrulation; it has also been seen in somite boundary during segmentation. (H–R) At later stages, chac1 becomes restricted to the forebrain, midbrain (MB), midbrain–hindbrain boundary (MHB), hindbrain (HB), telencephalon (Tel), retina and the myotome. It is also found in myotome, otic vesicle (OV), lens and heart at later stages of development. Scale bar: (E–H,N,Q), 100 µm; (I,J,L,M,O,P), 50 µm; (R), 25 µm.

Expression pattern of chac1 during zebrafish embryonic development

Chac1 was found to be strongly expressed during development. To determine the earliest time points of chac1 expression, we carried out real-time PCR analysis in zebrafish embryos at early stages of development. The chac1 mRNA was identified even at early stages of embryonic development beginning from 0.25 h post fertilization (hpf) to 6 hpf (Figure 1D). We subsequently carried out spatiotemporal expression profiling mRNA using whole-mount mRNA in situ hybridization. We observed chac1 expression as early as 0.25 hpf (Figure 1E,F) and its transcripts ubiquitously distributed up to 6 hpf and chac1 transcripts were also observed in EVL layer at 12 hpf (Figure 1G). In the later time points, chac1 transcripts were seen primarily in the developing brain, heart and myotome (Figure 1H–R).

chac1 expression could also be detected in regions of the developing eye where at 24 hpf it could be seen in the retina, but in the later time points, it seemed to be localized to the lens. We also observed chac1 expression in the otic vesicles representing the developing ear at later time points, and at these later time points (72 hpf) also significant expression in the heart became most apparent.

We also confirmed the spatiotemporal mRNA expression profiles by examining the protein expression through immunolocalization studies, where we observed a very similar pattern (Supplementary Figure SF2B).

Knockdown of chac1 leads to severe defects in development with eventual developmental arrest

To investigate the function of Chac1 in vertebrate embryogenesis, we carried out knockdown experiments with chac1-targeting morpholinos (chac1 MO) that were designed to block the translation by targeting the ATG start codon (Figure 2A). chac1 MO was injected into fertilized zebrafish embryo at the one- to two-cell stage and the phenotype was observed at different stages of the development (Supplementary Figure SF1A).

chac1 knockdown survival.

Figure 2.
chac1 knockdown survival.

(A) Schematic representation of coding sequence of zebrafish chac1 gene. Morpholinos were designed as indicated. (B) Bar plot of survivorship in percentage at different concentration of chac1MO. (C) Bar plot of survivorship at various developmental stages of chac1 morphant. (D) Efficiency test of morpholino treatment was inhibiting the expression of chac1. Protein extracts were prepared from WT, rescued and chac1MO-injected embryos at 48 hpf (long pectoral stage) subjected to Western blot analysis using human anti-CHAC1 antibody which recognizes zebrafish Chac1 and, anti-GAPDH antibody used as a control. Protein extracts from ∼15 embryos were loaded into each lane. Statistical comparisons are as indicated by (n = 80 embryos/group; n.s. not significant, *P < 0.05, **P < 0.001 and ***P < 0.0001, Student's t-test).

Figure 2.
chac1 knockdown survival.

(A) Schematic representation of coding sequence of zebrafish chac1 gene. Morpholinos were designed as indicated. (B) Bar plot of survivorship in percentage at different concentration of chac1MO. (C) Bar plot of survivorship at various developmental stages of chac1 morphant. (D) Efficiency test of morpholino treatment was inhibiting the expression of chac1. Protein extracts were prepared from WT, rescued and chac1MO-injected embryos at 48 hpf (long pectoral stage) subjected to Western blot analysis using human anti-CHAC1 antibody which recognizes zebrafish Chac1 and, anti-GAPDH antibody used as a control. Protein extracts from ∼15 embryos were loaded into each lane. Statistical comparisons are as indicated by (n = 80 embryos/group; n.s. not significant, *P < 0.05, **P < 0.001 and ***P < 0.0001, Student's t-test).

We screened the phenotypes using 100–500 µM concentration of chac1MO (Figure 2B,C). The 300 µM chac1MO produced the most balanced range of phenotypes. At this concentration, we observed considerable developmental defect at the initial stages of embryogenesis, followed by embryonic lethality, leading to an ∼83% death rate of embryos. We next examined protein abundance by western blot analysis in the chac1 morphant. No protein was detectable on immunoblots at 48 hpf indicating that there was indeed a successful block in translation (Figure 2D). To further assess the morphological changes in chac1 morphant, we performed microscopic examination. The microscopic examination of the zebrafish revealed delayed growth, lethality, curved tails, small eyes and pericardial edema in the chac1 morphant.

These results suggested that the chac1 gene has vital functions during embryonic development in zebrafish.

To ensure that the morpholino chac1MO was not leading to off-target effects, we used a second morpholino (chac1MO2) that targeted a different region (Figure 2A), and use of this morpholino reconfirmed the phenotypes (data not shown).

Knockdown of chac1 in zebrafish exhibit defects in organ development during embryogenesis

An examination of the chac1 morphant in more detail revealed severe defects in the myotome formation. The regular repeating patterns of the myotome seemed to be irregular in the morphant even at 24 hpf (Figure 3A). Hematoxylin and eosin (H& E) staining of the myotome of the 72 hpf morphant embryos demonstrated that although fiber formation was not affected, there was a disruption of muscle fiber arrangement. The fibers seemed disordered and contracted away from their sites of attachment compared with the WT (Figure 3B)

chac1 knockdown, its phenotypes and rescue.

Figure 3.
chac1 knockdown, its phenotypes and rescue.

(A) The morphology of the myotome was examined using a Stereomicroscope (Brightfield) in dechorionated embryos of 24 hpf (prim-5). Embryos were either injected with control MO (300 µM) or chac1MO (300 µM). Embryos were staged according to Kimmel et al. [19]. (B) Longitudinal sections of 72 hpf (protruding-mouth stage) embryos counterstained with hematoxylin and eosin stains showing the myotome trunk morphants with improper myofiber alignments. (C) The morphology of the brain was examined in dechorionated embryos of 24 hpf, and the loss of midbrain and hindbrain boundary is seen in chac1 morphants. (D) Heart of 72 hpf embryos; knockdown of chac1 caused heart defects, which show looping defects of morphants, the arrowed head indicates a large pericardial edema. (E) Bar plot showing the survivorship of rescued embryos with WT chac1 mRNA and catalytically inactive mutant of chac1 mRNA in percentage at 72 hpf (protruding-mouth stage). (F) The table represents the number of embryos that were used for knockdown and rescue experiments. Statistical comparisons are as indicated by (n = 80 embryos/group; n.s. not significant, **P < 0.001 and ***P < 0.0001, Student's t-test). Scale bar, 100 μm.

Figure 3.
chac1 knockdown, its phenotypes and rescue.

(A) The morphology of the myotome was examined using a Stereomicroscope (Brightfield) in dechorionated embryos of 24 hpf (prim-5). Embryos were either injected with control MO (300 µM) or chac1MO (300 µM). Embryos were staged according to Kimmel et al. [19]. (B) Longitudinal sections of 72 hpf (protruding-mouth stage) embryos counterstained with hematoxylin and eosin stains showing the myotome trunk morphants with improper myofiber alignments. (C) The morphology of the brain was examined in dechorionated embryos of 24 hpf, and the loss of midbrain and hindbrain boundary is seen in chac1 morphants. (D) Heart of 72 hpf embryos; knockdown of chac1 caused heart defects, which show looping defects of morphants, the arrowed head indicates a large pericardial edema. (E) Bar plot showing the survivorship of rescued embryos with WT chac1 mRNA and catalytically inactive mutant of chac1 mRNA in percentage at 72 hpf (protruding-mouth stage). (F) The table represents the number of embryos that were used for knockdown and rescue experiments. Statistical comparisons are as indicated by (n = 80 embryos/group; n.s. not significant, **P < 0.001 and ***P < 0.0001, Student's t-test). Scale bar, 100 μm.

In addition to this, there were clear defects observed in the morphant midbrain and hindbrain separation. While in the WT the midbrain and the hind brain could be clearly distinguished morphologically, this separation was absent from the chac1 morphant (Figure 3C).

Developmental defects in the heart were also observed. The WT heart was S-shaped and looped, In contrast, in the morphant, an elongated heart having a thin atrium and a compacted ventricle was observed with a massive pericardial edema in which the pericardium was also significantly distended possibly owing to the edema (Figure 3D). The heart was also significantly feebler with decreased heartbeats compared with the WT (Supplementary Video MS1A,B).

Together, these data demonstrate that the chac1 was crucial for the proper formation of the myotome, brain and heart during early development of zebrafish.

The chac1 knockdown phenotype can be rescued by chac1 mRNA but not by mRNA coding catalytically inactive Chac1

To examine whether the phenotypes being observed were specifically due to the chac1 knockdown, we carried out rescue experiments. We co-injected chac1MO and chac1 mRNA and observed that the embryonic lethality could be significantly attenuated by co-injecting with the chac1 mRNA. The survival increased dramatically from ∼11 to 55%. Furthermore, when we examined the specific organ defects, we observed that these could also be significantly rescued in the majority of zebrafish embryos (Figure 3E). To ensure that these were really rescued phenotypes, we only examined those embryos which also had significant levels of morpholinos injected as seen by the red fluorescence of the lissamine-tagged morpholinos (Supplementary Figure SF1B). These observations further confirmed that the phenotypes were specific to chac1 knockdown.

As a further proof of specificity, and to ensure that these were not merely off-target effects, we used an alternate control, where we repeated the rescue experiments using mRNA encoding the Chac1 (E95Q) mutation that led to a catalytically inactive protein. We observed that the survival levels were altered only marginally with the catalytically inactive chac1 mRNA (Figure 3E). The slight increase may be a consequence of the fact that in determining the survival frequencies, we evaluated all embryos and not just those that contained the morpholinos (indicated by the red fluorescence of the lissamine-tagged morpholinos). Taken together, these experiments establish the specificity of the chac1 knockdown phenotype and the dependence of the phenotype on the catalytic activity of Chac1 towards glutathione.

Zebrafish Chac1 expression leads to a change in the intracellular glutathione redox potential with a significant increase in the degree of oxidation

Although the zebrafish chac1 could be shown to alter the glutathione redox potential when it was heterologously expressed in yeast, it was important to determine whether Chac1 expression in the developing embryo was leading to an oxidizing environment in the developing embryos. We therefore evaluated the redox environment in the developing embryo in both WT and chac1 knockdown embryos. Grx1-roGFP2 has been recently shown to be able to measure the glutathione redox potential in adult zebrafish [31]. The Grx1-roGFP2 mRNA was prepared in vitro and injected into the one- to two-cell stage of the embryo. We evaluated the change in glutathione redox potential in the WT and the chac1 knockdown and observed that the cells expressing the Grx1-roGFP2 probe were more oxidizing in the WT cells where Chac1 was expressed. However, in chac1 knockdown cells, the glutathione redox potential of the cells was significantly more reducing (Figure 4A,B, Table 1 and Supplementary Figure SF2A) suggesting that Chac1 expression in these tissues was capable of creating a more oxidizing milieu even within the live animals.

Effect of Chac1 expression on intracellular glutathione redox potential of live zebrafish.

Figure 4.
Effect of Chac1 expression on intracellular glutathione redox potential of live zebrafish.

(A,B) Real-time intracellular glutathione redox potential mapping of zebrafish embryos expressing Grx1-roGFP2 at 10 hpf (bud; 100% epiboly stage) and 20 hpf (20-somite stage), respectively. In vivo time lapse images (time in second) show the redox state of chac1 morphant and WT, images were acquired by excitation at 405 and 488 nm and the corresponding ratio was taken. The rainbow color scale represents the level of redox state. Blue indicates a low level of redox state and yellow/white indicates a high level, normalized ratio values of morphant and WT embryos. Time is indicated as second in graphs. The orientation of intact embryos is lateral views. All statistical tests were performed in comparison with morphant and WT; experiments were performed with n = 82 embryos/group were analyzed and data represent the mean ± s.e.m. (n = 3). Scale bar: (A) 100 µm and (B) 200 µm, and all zoomed images; 50 µm.

Figure 4.
Effect of Chac1 expression on intracellular glutathione redox potential of live zebrafish.

(A,B) Real-time intracellular glutathione redox potential mapping of zebrafish embryos expressing Grx1-roGFP2 at 10 hpf (bud; 100% epiboly stage) and 20 hpf (20-somite stage), respectively. In vivo time lapse images (time in second) show the redox state of chac1 morphant and WT, images were acquired by excitation at 405 and 488 nm and the corresponding ratio was taken. The rainbow color scale represents the level of redox state. Blue indicates a low level of redox state and yellow/white indicates a high level, normalized ratio values of morphant and WT embryos. Time is indicated as second in graphs. The orientation of intact embryos is lateral views. All statistical tests were performed in comparison with morphant and WT; experiments were performed with n = 82 embryos/group were analyzed and data represent the mean ± s.e.m. (n = 3). Scale bar: (A) 100 µm and (B) 200 µm, and all zoomed images; 50 µm.

Table 1
Effect of chac1 on intracellular glutathione redox potential {EGSH (mV)}
Genotype 3 s 1 min 2 min 3 min 
1. chac1 overexpressed in yeast and corresponding empty vector 
WT yeast (vector) −328.70 −313.64 −313.54 −328.08 
chac1 o/v in yeast −263.18 −274.93 −263.52 −247.50 
2. Wild-type and chac1 morphants of zebrafish embryos 
chac1 morphant 10 hpf −303.14 −309.38 −307.00 −304.05 
WT 10 hpf −264.16 −263.30 −269.34 −271.93 
chac1 morphant 20 hpf −298.73 −301.45 −295.41 −293.18 
WT 20 hpf −266.77 −278.00 −273.71 −274.07 
Genotype 3 s 1 min 2 min 3 min 
1. chac1 overexpressed in yeast and corresponding empty vector 
WT yeast (vector) −328.70 −313.64 −313.54 −328.08 
chac1 o/v in yeast −263.18 −274.93 −263.52 −247.50 
2. Wild-type and chac1 morphants of zebrafish embryos 
chac1 morphant 10 hpf −303.14 −309.38 −307.00 −304.05 
WT 10 hpf −264.16 −263.30 −269.34 −271.93 
chac1 morphant 20 hpf −298.73 −301.45 −295.41 −293.18 
WT 20 hpf −266.77 −278.00 −273.71 −274.07 

Table showing change in intracellular glutathione redox potential compared to wild-type embryos, the knockdown of chac1 showed less intracellular redox potential (EGSH) in both 10 hpf (bud; 100% epiboly stage) and 20 hpf (20-somite stage) whereas overexpression of chac1 in yeast vector was showing higher intracellular redox potential compared with empty vector (control vector).

The chac1 knockdown leads to a strong attenuation of calcium transients in the developing zebrafish embryo

We have previously observed that the expression of Chac1 proteins can alter the intracellular glutathione redox potential in yeast and lead to activation of calcium channels [17,18]. We needed to assess, therefore, the effect of chac1 knockdown on calcium signals in the developing zebrafish. We injected GCaMP6s mRNA with or without chac1 morpholino and examined the calcium signals in vivo. The GCaMP6s was expected to be expressed in all the tissues but significant levels of proteins were only expected to be detectable at later time points (since only with the use of transgenic GCaMP6s lines could calcium be detected) at the early time points [32]. Indeed, the early time points (3 and 6 hpf) did not reveal any detectable signal. However, from 10 hpf onwards, we could clearly detect the calcium transients in the WT zebrafish embryos in the EVL layer. A time-lapse video of the EVL layer is shown revealing the calcium spikes (Figure 5A and Supplementary Video MS2A). Interestingly, these calcium spikes or transients were dramatically attenuated in the chac1 knockdown (Figure 5B and Supplementary Video MS2B). We compared the signal intensities and a significant difference could be observed between WT and chac1 knockdown. A similar observation was observed at the 20 and 26 hpf time points where the signal intensities in the live zebrafish are reflective of the myotome and the MHB layers (Figure 5C,D and Figure 6A,B). Although this difference was also discernible at the 48 hpf, the probe signal had become much weaker at this time point. These results clearly demonstrate that calcium transients were dependent on the Chac1 expression in these tissues.

Live calcium imaging in the WT and chac1 morphants of 10 and 20 hpf

Figure 5.
Live calcium imaging in the WT and chac1 morphants of 10 and 20 hpf

The GCaMP6s mRNA was injected at one- to two-cell stage and time series images were acquired using confocal microscopy. The images were acquired from animal pole every 7.5 s interval up to 3 min. Mean values for fluorescence at 488 nm were monitored over time in 10 distinct ROIs (n = 3), in response to calcium signaling in wild type and chac1 morphant. (A,B) Calcium signal in EVL region at 10 hpf (bud; 100% epiboly stage). The orientation of embryos was lateral view with the dorsal side at the right and the anterior towards the top (with few angles indicated by arrow heads) for (A) and similarly, for (B), but the anterior at the top (with few angle indicated by arrow heads). (C,D) Calcium signal in brain and myotome region at 20 hpf (20-somite stage). The orientation of embryos were lateral views, anterior toward the left, dorsal towards the top. All statistical tests were performed in comparison with morphant and WT; experiments were performed with n = 82 embryos/group were analyzed and data represent the mean ± s.e.m. (n = 3). P < 0.001 was considered to be significant and indicated by ‘**’, Student's t-test. Scale bar: 100 µm and all zoomed images; 50 µm.

Figure 5.
Live calcium imaging in the WT and chac1 morphants of 10 and 20 hpf

The GCaMP6s mRNA was injected at one- to two-cell stage and time series images were acquired using confocal microscopy. The images were acquired from animal pole every 7.5 s interval up to 3 min. Mean values for fluorescence at 488 nm were monitored over time in 10 distinct ROIs (n = 3), in response to calcium signaling in wild type and chac1 morphant. (A,B) Calcium signal in EVL region at 10 hpf (bud; 100% epiboly stage). The orientation of embryos was lateral view with the dorsal side at the right and the anterior towards the top (with few angles indicated by arrow heads) for (A) and similarly, for (B), but the anterior at the top (with few angle indicated by arrow heads). (C,D) Calcium signal in brain and myotome region at 20 hpf (20-somite stage). The orientation of embryos were lateral views, anterior toward the left, dorsal towards the top. All statistical tests were performed in comparison with morphant and WT; experiments were performed with n = 82 embryos/group were analyzed and data represent the mean ± s.e.m. (n = 3). P < 0.001 was considered to be significant and indicated by ‘**’, Student's t-test. Scale bar: 100 µm and all zoomed images; 50 µm.

Live calcium imaging in the WT and chac1 morphants of 26 hpf

Figure 6.
Live calcium imaging in the WT and chac1 morphants of 26 hpf

(A,B) in midbrain–hindbrain boundary region at 26 hpf (prim-6 stage). Magnification, 10×. The orientation of embryos was dorsal view. All statistical tests were performed in comparison with morphant and WT; experiments were performed with n = 82 embryos/group were analyzed and data represent the mean ± s.e.m. (n = 3). P < 0.001 was considered to be significant and indicated by ‘**’, Student's t-test. Scale bar: 100 µm and all zoomed images; 20 µm.

Figure 6.
Live calcium imaging in the WT and chac1 morphants of 26 hpf

(A,B) in midbrain–hindbrain boundary region at 26 hpf (prim-6 stage). Magnification, 10×. The orientation of embryos was dorsal view. All statistical tests were performed in comparison with morphant and WT; experiments were performed with n = 82 embryos/group were analyzed and data represent the mean ± s.e.m. (n = 3). P < 0.001 was considered to be significant and indicated by ‘**’, Student's t-test. Scale bar: 100 µm and all zoomed images; 20 µm.

Discussion

We describe in this manuscript the critical role played by the intracellular glutathione redox potential in the development of a vertebrate animal such as the zebrafish. Previous studies have speculated that there is likely to be a role for redox during development. In these earlier studies with zebrafish, the authors had shown how glutathione-related enzymes alter their expression in the developing embryo [33,34]. These studies evaluated the expression of a large number of enzymes involved in glutathione homeostasis but did not include ChaC1, probably because the role of ChaC1 in glutathione degradation had only just been established at the time these studies emerged [14]. Using measurements of reduced and oxidized glutathione in whole embryos, these authors also attempted to determine the overall change in the glutathione redox potential during development and observed an increase in the oxidizing potential up to 12 hpf followed by a shift to a more reducing potential in an oscillating manner. Despite the significant amount of work carried out in these studies, one drawback has been that the glutathione redox potential was estimated in whole embryos that included multiple developing tissue types. However, redox changes are dynamic processes, can vary with cell types and thus correlation with whole embryos are likely to be less informative, and sometimes, misleading. Thus, using the Grx1-roGFP2 probe, we could observe that the glutathione redox potential increased even at 72 hpf, but only in specific developing organs, and that in the majority of the embryo, the glutathione redox potential did not change. This underlines the need for genetically encoded redox indicators for studying these processes in complex tissues or in developing embryos. Furthermore, tools required for altering the redox potential in a controlled manner during development are also required but have not been available. This is because ROS-generated or H2O2 generated redox changes are not easy to control in a developing animal. Thus, the recently described ChaC1 protein appears to be a unique mechanism available to the organism to regulate its glutathione redox potential in a precise manner. We have further shown that the increase in the oxidation status of the cells through the alteration of the glutathione redox potential triggers calcium release, a factor known to be critical for the development. It is interesting to note that the myotome, brain and heart, where calcium has been reported to have a clear role in development, are in fact the organs where chac1 was principally expressed and where the chac1 knockdown phenotypes were the most discernible. Interestingly, the chac1 was also identified in a screen for genes required for neurogenesis in mouse [35], and based on our findings here, it is likely that it would also play a role in the muscle and heart development of the mouse as well.

It is interesting that while the biosynthesis of glutathione is essential for embryonic development [36], the degradation of glutathione is also essential for development, as shown in this study. In the latter case, however, it is likely to be the altered glutathione redox potential (that results from Chac1 degradation of reduced glutathione) that is the critical element, since in the presence of ongoing biosynthesis, the alteration in overall glutathione levels itself is not that significant. In previous studies with the membrane-bound glutathione degrading enzyme, ϒ-GT which acts on external pools of both the oxidized and reduced forms of glutathione, the knockout of this enzyme was not found to be lethal [37]. Two important differences with Chac1-mediated degradation, is that Chac1 acts on cytoplasmic pools, and that its primary effect is the ability to alter the redox potential that serves an activation signal.

Although our results reveal the role of calcium release that occurs following on Chac1-mediated alteration in the glutathione redox potential, we have not been able to pinpoint the precise calcium channels that might be activated by the altered glutathione redox potential. There are a large number of calcium channels encoded in the zebrafish genome, and more work would be required to determine the tissue specificities and localizations of these channels, and furthermore to determine which of them may in fact be regulated by the glutathione redox potential.

The previously known activators of calcium stores are the metabolites IP3, cADPR and NAADP [810]. IP3 and cADPR act at the ER through the Ryanodine family of receptors, while NAADP acts to release calcium stores of the endolysosomal system through the two-pore channels. Both the Ryanodine receptors [11] and the two-pore channels are known to be regulated by a variety of other factors [12,13] and in the case of the Ryanodine receptors, this also includes redox [29]. These channels have a demonstrated role in myofibril bundling and alignment in the slow muscles, and blocking either channel affects the musculature of the developing zebrafish [3840], similar to what is observed in the chac1 knockdown cells. In the present study, we have not identified whether these or other redox-sensitive calcium channels were being activated upon the alteration in the glutathione redox potential during Chac1 expression. An exciting avenue for future work is to not only to delineate these channels but to also determine whether redox has a consequence on the levels of other metabolite effectors of calcium mobilization or of other pathways that might influence or be impacted by calcium mobilization [35,41,42]. Thus, many intriguing possibilities open up with the recognition that the glutathione redox potential is one of the upstream activators of calcium mobilization during development, and it is likely that Chac1 would be a powerful tool in investigating these questions.

Abbreviations

     
  • γ-GT

    γ-glutamyl transpeptidase

  •  
  • DTT

    dithiothreitol

  •  
  • MHB

    midbrain–hindbrain boundary

  •  
  • MOs

    morpholino oligonucleotides

  •  
  • PFA

    paraformaldehyde

  •  
  • ROI

    region of interest

  •  
  • RT

    room temperature

Author contribution

A.K.B. and S.Y. conceived the project, A.K.B, S.Y. and R.R. designed and planned the experiments, S.Y. executed majority of the experiments, B.C. performed the whole-mount mRNA in situ hybridization, M.A.K. performed qRT-PCR and mRNA and cDNA preparation from zebrafish for qRT-PCR, R.R. performed chac1MO microinjections, A.K.B., R.R. and S.Y. analyzed and interpreted data, S.Y. processed and illustrated all images. S.Y. and A.K.B. wrote the manuscript, and A.K.B. was involved in the overall supervision of the project.

Funding

S.Y. acknowledges a fellowship from DST INSPIRE [No. IF150103]. B.C. acknowledges a fellowship from CSIR. M.A.K. acknowledges a fellowship from IISER Mohali. R.R. was a recipient of the Wellcome Trust/DBT India Alliance Intermediate Fellowship [IA/I/12/2/500630]. R.R. also acknowledges extramural research funding from Department of Biotechnology, Government of India [102/IFD/SAN/3975/2015-2016] and [102/IFD/SAN/2255/2017-2018]. A.K.B. was a recipient of a JC Bose National Fellowship (2012–2017) from the Department of Science and Technology, Government of India. This work was funded by a grant in aid project to A.K.B. [Project No. SB/SO/BB/017/2014].

Acknowledgements

We thank Dr Saikat Ghosh and Devashish Dwivedi for help with the microscopy and imaging, Dr Avinash Chandel for help with the Grx-roGFP2 experiments, Dr Poonam Sharma for help in the in situ hybridization.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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