2-Deoxy-D-glucose (2-DG) treatment of embryos

2-Deoxy-D-glucose (2-DG) Treatment of Zebrafish Embryos

Activation of the Mondo Pathway via Microinjection

1. Introduction and Principle

This protocol describes the use of the glucose analog 2-deoxy-D-glucose (2-DG) to selectively activate the Mondo pathway in zebrafish embryos via microinjection. The method exploits the metabolic properties of 2-DG to generate a specific intracellular signal without engaging downstream glycolytic pathways.

1.1 Scientific Rationale

Glucose-6-phosphate (G6P) is the intracellular signal thought to activate the MondoA/MLX transcription factor complex. When glucose enters cells, hexokinase phosphorylates it to G6P, which then proceeds through glycolysis and other metabolic routes. 2-DG is a glucose analog in which the hydroxyl group at the C-2 position is replaced by hydrogen. Hexokinase phosphorylates 2-DG to 2-deoxy-D-glucose-6-phosphate (2-DG-6P), but this product cannot be further metabolized by phosphoglucose isomerase, and therefore accumulates intracellularly.

This accumulation of 2-DG-6P mimics the G6P signal and activates the Mondo pathway, while avoiding activation of other pathways that depend on further glucose metabolism [1-3].

1.2 Experimental Applications Covered

This protocol covers two distinct experimental paradigms:

• Application A: In vivo bioluminescence recording from a ChoRE (Carbohydrate Response Element) reporter following 2-DG stimulation.

• Application B: RT-qPCR analysis to determine whether glucose induction of target gene expression (e.g., txnipa) is dependent on MondoA, by co-injection of 2-DG with mondoa-mo or mondoa-mis morpholinos.

   

2. Materials and Reagents

2.1 Chemicals and Reagents

2.2 Plasticware and Consumables (for Bioluminescence)

2.3 Biological Reagents

2.4 Equipment

• Gas-driven microinjector: Eppendorf FemtoJet express. Alternatives include FemtoJet 4i/4x or PicoSpritzer III.
• Borosilicate glass capillary tubes with internal filament (~0.6 mm inner diameter, ~1.0 mm outer diameter; e.g., Warner Instruments, Hamden, USA).
• Micropipette puller: Sutter P-97 Flaming (Sutter Instrument Co., Novato, USA). Optimise pulling parameters for a needle with moderate taper length (~8 mm cone).
• Microloader tips (e.g., Eppendorf Microloader) for back-filling needles.
• Stereomicroscope with transmitted light base (e.g., Leica, Zeiss Stemi, or Nikon SMZ series).
• Stage micrometer (0.01 mm divisions; e.g., Fisher 12-561-SM1) for droplet volume calibration.
• Mineral oil (e.g., Sigma-Aldrich cat. no. M8662 or any other mineral oil) for injection volume calibration.
• Fine forceps (e.g., Dumont #5) for breaking needle tips and dechorionation.
• 96-well white-walled plates (for bioluminescence; e.g., OptiPlate-96 white flat-bottom).
• Plate-reading luminometer: PerkinElmer EnVision XCite Multilabel Plate Reader with enhanced luminescence detector. Alternatives: PerkinElmer Topcount NXT, Promega GloMax 9, or Tecan Infinite M200.
• Incubator set at 28.5°C.
• Micropipettes and filter tips.

3. Solution Preparation

3.1 2-DG Stock Solution (300 mM)

1. Calculate the required mass of 2-DG. Molecular weight of 2-DG = 164.16 g/mol. For a 300 mM solution in 1 ml: weigh 49.2 mg of 2-DG.

2. Dissolve in 1 ml nuclease-free water by gentle vortexing.

3. Filter-sterilize through a 0.22 µm syringe filter if desired.

4. Prepare aliquots of 10-20 µl to avoid repeated freeze-thaw cycles.

5. Store aliquots at -20°C. Thaw on ice immediately before use.

Note: The 300 mM concentration is the injection mix concentration. The actual intracellular concentration will be substantially lower due to dilution within the embryo.

3.2 Luciferin Stock Solution (50 mM)

Prepare the luciferin stock solution:

1. Prepare a 50 mM aqueous luciferin stock by adding distilled H₂O to the vial containing D-Luciferin Firefly, potassium salt (Biosynth, L-8220). For example, dissolve 1 g of luciferin powder in 62.8 ml dH₂O.

2. Aliquot the stock solution into light-protected tubes (e.g., amber or foil-wrapped 1.5 ml tubes).

3. Store aliquots at -80°C. The stock is stable for several months at -80°C.

Caution: D-luciferin is light-sensitive. Minimise light exposure during preparation, aliquoting, and use. Work quickly and keep tubes covered.

3.3 E3 Medium with Luciferin (E3L)

Prepare E3L medium fresh on the day of the experiment:

1. Prepare standard E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, 0.33 mM MgSO₄).

2. Thaw an aliquot of 50 mM luciferin stock on ice, protected from light.

3. Dilute the luciferin stock into E3 medium to a final concentration of 0.5 mM. For example, add 500 µl of 50 mM luciferin stock to 50 ml E3 medium.

4. Protect from light by wrapping tubes/plates in aluminium foil. Use freshly prepared E3L on the same day.

3.4 Injection Mixes

Prepare injection mixes on ice immediately before injection. All volumes are approximate and should be scaled as needed. A typical total injection mix volume is 5-10 µl.

Application A: ChoRE Reporter Bioluminescence Assay

Application B: mondoa Knockdown + 2-DG (txnipa RT-qPCR)

Note: Phenol red (0.1% final) may be added to all mixes as a visual injection tracer, unless it interferes with downstream readouts.

4. Procedure

4.1 Needle Preparation

1. Pull borosilicate glass capillary tubes (~1.0 mm OD, ~0.6 mm ID, with internal filament) using the Sutter P-97 Flaming/Brown Micropipette Puller (or Narishige PC-10). Use established pulling parameters for your instrument to produce needles with a moderate taper (~8 mm cone length). Each pull produces two needles.

2. Store pulled needles on a strip of adhesive putty or modelling clay inside a covered Petri dish to prevent dust accumulation and breakage.

3. Under the stereomicroscope, break the closed tip of the needle using fine forceps (Dumont #5) to create an opening of approximately 5–10 µm. The opening should be large enough to allow fluid ejection without excessive leakage.

4. Back-fill the needle with 2–3 µl of injection mix using a microloader tip (e.g., Eppendorf Microloader). Insert the microloader to near the needle tip and slowly dispense the solution; the internal filament will wick the solution to the tip.

Note: Pull more needles than needed before starting injections. This avoids delays if a needle breaks or clogs during the experiment.

4.2 Injection Volume Calibration (Mineral Oil Method)

Accurate calibration of injection volume is critical for reproducibility. The following procedure uses the mineral oil droplet method to measure and adjust the volume delivered per injection pulse.

4. Place a small drop (approximately 5–10 µl) of halocarbon oil (Sigma H8773) or mineral oil on a stage micrometer (0.01 mm scale divisions). The oil provides a hydrophobic medium in which the aqueous injection bolus forms a discrete, measurable sphere.

5. Mount the loaded needle onto the Eppendorf FemtoJet express microinjector. Set initial injection parameters: injection pressure (Pi) approximately 100-300 hPa, injection time (ti) approximately 0.1-0.5 seconds, and compensation pressure (Pc) approximately 15-30 hPa. These are starting values and will be adjusted.

6. Open the needle carefully with your forceps.

7. Position the needle tip within the oil drop under the stereomicroscope. Trigger one injection pulse using the foot pedal. A small sphere of injection solution (visible due to phenol red) will form in the oil.

8. Measure the diameter (d) of the sphere using the stage micrometer scale. Read the diameter in micrometer divisions.

9. Calculate the droplet volume using the formula for the volume of a sphere: V = (π/6) × d³. For example, a sphere with a diameter of 0.125 mm (125 µm) has a volume of approximately 1.0 nl.

Quick reference for common droplet diameters:

10. If the droplet volume is too large or too small, adjust the injection pressure (Pi) and/or injection time (ti) on the FemtoJet and repeat the calibration. Reduce pressure/time for smaller volumes; increase for larger.

11. Once the desired volume (typically 1-2 nl) is reproducibly achieved across 3-5 consecutive test pulses, the needle is calibrated and ready for embryo injection.

12. Recalibrate the needle if it becomes clogged and is re-opened, or if a new needle is loaded.

Caution: The compensation pressure (Pc) prevents backflow of medium into the needle tip. If set too high, it causes continuous leakage; if too low, the solution may be diluted by capillary backflow. Adjust Pc so that the meniscus at the needle tip remains stable with no visible dripping.

Note: Record the calibrated injection parameters (Pi, ti, Pc) and needle tip diameter for each experiment in your lab notebook to aid reproducibility.

4.3 Embryo Collection and Preparation (Semi-Dry Injection Method)

This protocol uses a semi-dry injection technique (but this is a matter of personal preference): embryos are positioned in minimal or no medium during injection and E3 is added back immediately afterwards. Removing the surrounding liquid prevents embryos from floating or rolling during needle insertion and improves injection precision.

1. Set up zebrafish breeding pairs the evening before the experiment. Use standard crossing protocols appropriate for your facility.

2. Collect embryos within 15-20 minutes of fertilisation to ensure injection at the one-cell (zygote) stage.

3. Transfer embryos briefly into a Petri dish containing E3 medium. Remove any unfertilized or damaged eggs.

4. Using a plastic Pasteur pipette or fine-tip transfer pipette, carefully remove as much E3 medium as possible from around the embryos. Leave only a thin film of moisture so that the embryos do not dry out but are not submerged. The embryos should sit on the bottom of the petri dish without floating or moving.

Note: Do not leave embryos completely dry for extended periods. The semi-dry condition should be maintained only during the injection session. Work quickly through each batch and add E3 back promptly after injection (see Section 4.4).

4.4 Microinjection into Zygotes (Semi-Dry)

Embryos should already be arranged (see Section 4.3). The semi-dry condition keeps embryos stationary and prevents the needle tip from being deflected by surrounding liquid.

1. Pierce the chorion and cell membrane with the needle tip, targeting the interface between the yolk cell and the blastomere.

2. Deliver the calibrated volume (1-2 nl) by triggering the FemtoJet via the foot pedal. Confirm successful injection by observing the phenol red bolus within the cell/yolk.

3. Immediately after completing injection of each batch, gently add E3 medium back to the dish to re-submerge the embryos.

4. Incubate embryos at 28.5°C until the desired developmental stage.

5. At 2-4 hours post-injection, assess embryos under the stereomicroscope and remove any dead or abnormal embryos. Record survival rates.

Caution: Ensure injection occurs at the one-cell (zygote) stage for uniform distribution of injected material. The first cleavage occurs approximately 15–20 minutes after fertilization; injection into later-stage embryos may lead to mosaic distribution.

Caution: Do not leave embryos in the semi-dry state for more than a few minutes. Prolonged exposure without E3 can cause desiccation and increased mortality. Add E3 back as soon as each batch of injections is completed.

5. Application-Specific Downstream Procedures

5.1 Application A: In Vivo Bioluminescence (ChoRE Reporter)

This procedure measures Mondo pathway activation in live embryos using a 2xChoRE-driven luciferase reporter (see also [5, 6]).

1. At high/oblong stage (approximately 3.3 hpf), select healthy injected embryos.

2. Using a wide-bore 1 ml pipette tip (cut ~5 mm from the end and briefly flame the sharp edges), transfer individual embryos into the wells of a white opaque 96-well plate (OptiPlate-96, PerkinElmer, cat. 6005299). Each well should contain 225 µl E3L medium (E3 + 0.5 mM luciferin; see Section 3.3).

3. Include wells with E3L only (no embryo) as background controls for subtraction of non-specific luminescence. Reserve at least 4–8 background wells per plate.

4. Seal the plate with TopSeal-A adhesive transparent sealing sheets (PerkinElmer, cat. 6005185) to prevent evaporation while allowing light transmission for luminescence detection.

5. Measure bioluminescence at sphere stage (approximately 4 hpf) using the plate-reading luminometer at 28°C.

Recommended luminometer settings (PerkinElmer EnVision with enhanced luminescence detector):

6. Subtract the mean background luminescence (from embryo-free wells) from all embryo readings.

Note: Maintain consistent timing between injection, plating, and measurement across all groups. Stagger injections if necessary to synchronize developmental staging at readout.

Note: If signal is too low: we recommend pooling ~20 larvae and measuring reporter activity using an in vitro luciferase assay (Promega Luciferase Assay System, cat. E1500) as an endpoint measurement. Homogenise embryos in Reporter Lysis Buffer (Promega, cat. E3971) using micropistilles (Eppendorf, cat. 0030120.973) and pass the lysate through a syringe (0.45 × 25 mm, Braun Sterican) four times before assaying.

5.2 Application B: RT-qPCR for txnipa (mondoa Knockdown)

This procedure tests whether glucose induction of txnipa expression is dependent on MondoA function, by comparing 2-DG-treated embryos injected with mondoa-mo versus the mondoa-mis mismatch control (Weger et al., 2020 [7], Figure 1-figure supplement 1G).

• Inject zygotes with 150 mM 2-DG or water along with either mondoa-mo or mondoa-mis at the validated dose (four conditions total: mondoa-mo + 2-DG, mondoa-mo + water, mondoa-mis + 2-DG, mondoa-mis + water; see Section 3.4).
• Incubate injected embryos at 28.5°C until sphere stage (approximately 4 hpf).
• Collect embryos in pools into 1.5 ml tubes. Remove as much E3 medium as possible.
• Snap-freeze in liquid nitrogen or add RNA extraction buffer (e.g., TRIzol) immediately.
• Extract total RNA using a standard protocol (TRIzol/chloroform or column-based kit). Assess RNA quality and quantity by spectrophotometry and Bioanalyzer or electrophoresis.
• Perform reverse transcription using a standard cDNA synthesis kit with oligo-dT and/or random hexamer primers.
• Perform qPCR against txnipa using validated primers. Include appropriate reference genes (e.g., ef1α).
• Analyse using the ΔΔCt method or equivalent. Compare txnipa expression across all four conditions. The critical comparison is mondoa-mo + 2-DG versus mondoa-mis + 2-DG: if glucose induction of txnipa is abolished by mondoa-mo but present with mondoa-mis, this confirms MondoA dependence (n = 3 biological replicates, >20 embryos each).

7. Developmental Staging Reference

Precise staging is critical to this protocol. All staging follows the standard zebrafish staging series [8].

8. References

1. Li, M.V., et al., Glucose-6-phosphate mediates activation of the carbohydrate responsive binding protein (ChREBP). Biochem Biophys Res Commun, 2010. 395(3): p. 395-400.  

2. Stoltzman, C.A., et al., Glucose sensing by MondoA:Mlx complexes: a role for hexokinases and direct regulation of thioredoxin-interacting protein expression. Proc Natl Acad Sci U S A, 2008. 105(19): p. 6912-7.

 3. Li, M.V., et al., Glucose-dependent transcriptional regulation by an evolutionarily conserved glucose-sensing module. Diabetes, 2006. 55(5): p. 1179-89.

 4. Nüsslein-Volhard, C., ed. Zebrafish. A Practical Approach. Vol. 261. 2002, Oxford University Press: Oxford.

 5. Weger, B.D., et al., A Chemical Screening System for Glucocorticoid Stress Hormone Signaling in an Intact Vertebrate. ACS Chem Biol, 2012. 7(7): p. 1178-83.

6. Weger, B.D., et al., A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System. J Vis Exp, 2013: p. e50439.

7. Weger, M., et al., MondoA regulates gene expression in cholesterol biosynthesis-associated pathways required for zebrafish epiboly. Elife, 2020. 9.

8. Kimmel, C.B., et al., Stages of embryonic development of the zebrafish. Dev Dyn, 1995. 203(3): p. 253-310.