In Vivo Modulation and Quantification of microRNAs During Axolotl Tail Regeneration

Abstract

The ability to regenerate diseased, injured, or missing complex tissue is widespread throughout lower vertebrates and invertebrates; however, our knowledge of the molecular mechanisms that regulate this amazing ability is still in its infancy. Many recent papers have shown important roles for microRNAs in regulating regeneration in a number of species. The ability to detect and quantify miRNA expression fluctuations at a single cell level in vivo in different cell types during processes like regeneration is very informative. In this chapter, we describe how to use a dual-fluorescent green fluorescent protein (GFP)-reporter/monomeric red fluorescent protein (mRFP)-sensor (DFRS) plasmid to quantitate the dynamics of specific miRNAs over time following miRNA mimic injection as well as during regeneration. In this bicistronic vector, the mRFP allows for verification of miRNA expression, while the GFP functions as an internal control to normalize miRNA expression and thus obtain quantitative results. In addition, we demonstrate how this technique revealed dynamic miR-23a expression and function during tail regeneration.

1 Introduction

The ability to regenerate multiple body parts is a fascinating phenomenon that remains poorly understood at a molecular level [1–12]. While model systems like axolotls are desirable since they can regenerate these tissues, lack of a full axolotl genome sequence hinders classical genetic studies. Recent studies have shown that small noncoding microRNAs (miRNAs) are highly conserved and may play an important role in regulating gene expression after injury in many species including axolotls [13–19].

miRNAs are small noncoding RNAs that are approximately 22 nucleotides long [20–22]. The major function of miRNAs is post-transcriptional regulation of gene expression. The canonical mechanism of action for miRNAs is to bind to a region of complementary sequence in the 3′ untranslated region (UTR) of the target messenger RNA (mRNA). The binding of the miRNA to its target mRNA often leads to message degradation [23]. miRNAs play an essential role in regeneration [19, 24–26], yet dynamics of miRNA expression during regeneration remains to be delineated. One tool that allows us to explore temporal fluctuation of specific miRNAs in a tissue of interest is the dual-fluorescent green fluorescent protein (GFP)-reporter/monomeric red fluorescent protein (mRFP)-sensor (DFRS) plasmid (Fig. 1) [27, 28]. In addition to normal bacterial expression components, the DFRS plasmid is a bicistronic GFP (reporter) and mRFP (sensor) transiently expressed under the control of two independent SV40 promoters. Sequences that are complementary to an miRNA of interest can be cloned into the 3′UTR of mRFP in the DFRS plasmid using a variety of restriction enzymes for digestion followed by ligation [21].

Fig. 1.

Schematic of DFRS plasmid. This dual miRNA reporter construct can be used to analyze and quantify expression of endogenous miRNAs and to quantitatively evaluate the effect of modulators of microRNAs, like inhibitors or mimics. The DFRS plasmid contains both GFP (control) and mRFP (reporter), each under the control of their own SV40 promoter. Following the open read frame of mRFP is a multiple cloning site (MCS) to allow addition of seed sequences for an miRNA of interest

Using microinjection and electroporation, the DFRS plasmid can be injected into a tissue of interest. The expression of the DFRS plasmid will not only confirm miRNA mimic uptake into a specific cell but will also allow observation of changes in expression levels of miRNA in a single cell in vivo. By live animal imaging, the amount of mRFP can be normalized to GFP, with the changes in mRFP negatively correlate with activity of the given miRNA, such that the relative decrease in mRFP expression correlates with increased miRNA expression in that cell. This can be used to model temporal changes in expression of an miRNA of interest as regeneration progresses as well as spatial changes as miRNA expression fluctuates relative to the regenerating tissue.

2 Materials

1. Fluorescent dissecting scope.

2. Pressure injector.

3. Micromanipulator.

4. Electroporator.

5. Electrodes.

6. Needle puller.

7. Axolotls.

8. miRNA DFRS plasmids.

9. miRNA mimic.

10. Anesthesia (0.01 % p-aminobenzene).

11. Fast Green.

12. Glass capillaries.

13. Sylgard-dissecting pad.

14. Insect pins.

15. Microloader pipette tips.

16. 1× PBS.

17. Tweezers.

18. Image J.

3 Methods

3.1 Preparation of Plasmid Solution for Injection

1. To prepare the plasmid solution for injection, dilute the dual-fluorescent green fluorescent protein (GFP)-reporter/monomeric red fluorescent protein (mRFP)-sensor (DFRS) plasmid to a final concentration of 0.5 μg/μL with 0.2 mg/mL Fast Green and 1× phosphate buffered saline (PBS) (see Note 1).

3.2 Preparation of Needles

1. Pull glass capillaries to form injection needles (see Note 2). Following a ramp test, place a glass capillary into the needle puller and input the following settings: Heat (Ramp value + 10), Pull 60, Velocity 60, Time 250, and Pressure 500.

2. Load 2–4 μL of plasmid solution from Subheading 3.1 into the injection needle using a pipette with a microloader tip.

3. Insert and secure the loaded injection needle into the pressure injector nozzle attached to the micromanipulator.

4. To open the needle, very gently break the very tip using a forceps. To ensure the needle is open, press the injection pedal to observe release of the plasmid solution.

3.3 Injection and Electroporation of Animals

1. Set the pressure injector to 20 psi ejection pressure and 100 ms timed ejection.

2. Place axolotls into anesthesia (see Note 3).

3. Once the axolotl is motionless and unresponsive to touch, place the axolotl on a Sylgard-dissecting pad and stabilize the axolotl by pinning the fin down with four insect pins. Assure the axolotl remains moist by adding a drop of the anesthetic on the axolotl’s head.

4. Visualize the target tissue using a dissecting microscope.

5. Using the micromanipulator, insert the injection needle into the tissue of interest, such as muscle or spinal cord, and press the injection pedal to release the plasmid solution (see Note 4).

6. While the axolotl is still pinned on the Sylgard-dissecting pad, cover the axolotl with 1× PBS. Place the electrodes on either side of the tail ensuring that either electrode is not in direct contact with tissue.

7. Electroporate the axolotl with 5 pulses of 50 V, 50 ms each (see Note 5).

8. Remove the insect pins and return the axolotl to water (see Note 6).

3.4 Tail Amputation

1. Place axolotls into anesthesia (see Note 7).

2. Once the axolotl is motionless, place the axolotl in a petri dish under a fluorescent dissecting microscope.

3. Visualize the DFRS plasmid-expressing cells by GFP expression. Align a scalpel immediately posterior to GFP-expressing cells. Amputate the tail using a scalpel.

4. Image the axolotl about the amputation, and return to water.

5. Image the axolotl at desired time points during regeneration. Be sure to use the same exposure time as well as other microscope settings as the first imaged time point (see Note 8).

3.5 Mimic Injection

1. Prepare the mimic solution so the final concentration of the miRNA mimic is 10 μM with 0.2 mg/mL Fast Green and 1× PBS (see Notes 9–11).

2. Load a new injection needle with 2–4 μL of mimic solution using a pipette with a microloader tip. Insert and secure the needle into the pressure injector nozzle attached to the micromanipulator. To open the needle, very gently break the very tip using a forceps. To ensure the needle is open, press the injection pedal to observe release of the plasmid solution.

3. Place axolotls into anesthesia.

4. Once the axolotl is motionless and unresponsive to touch, place the axolotl on a Sylgard-dissecting pad and stabilize the axolotl by pinning the fin down with four insect pins. Assure the axolotl remains moist by adding a drop of the anesthetic on the axolotl’s head.

5. Place the Sylgard-dissecting pad under a fluorescent dissecting scope. Visualize the DFRS plasmid-expressing cells by GFP expression, and adjust the position of the needle to inject immediately adjacent to the DFRS plasmid-expressing cells as to not damage the cell by puncture.

6. Release the mimic solution by pressing the pedal of the injector using the settings in Subheading 3.1 (see Note 12).

7. Cover the axolotl in 1× PBS and electroporate as in Subheading 3.3.

8. Remove the pins and image the injected area of the axolotl (Fig. 3a).

9. Return the axolotl to water.

10. Image the axolotl at desired time points (see Note 13).

Fig. 3.

Determining the effect of microRNA mimic injection using miR-141a sensor. (a) Control GFP expression (green) and reporter mRFP expression (red) shown at 0, 24, 48, and 96 hour post mimic injection (hpi). Arrowheads indicate examples of cells that are expressing the DFRS plasmid and are used for quantification. Scale bar is 200 μm. (b) Graphical representation of mRFP expression modulated by miR-141a in the spinal cord following mimic injection. Overall there is a general decrease in the amount of mRFP expression until 96 hpi, indicating that most DFRS plasmid-expressing cells also took up and processed injected miR-141 mimics (***P < 0.001, **P < 0.01, NS not significant)

3.6 Quantification

1. Open RFP and GFP channel images in separate windows in image J (or similar program).

2. Synchronize windows by going to Analyze →Tools→Synchronize Windows →Synchronize All.

3. Select the freehand selection tool and trace the outline of the DFRS plasmid-expressing cell.

4. Measure the fluorescence intensity of the GFP window by going to Analyze → Measure. Be sure the measurement output contains “Area,” “Integrated Density,” and “Mean.” Repeat the measurement on the RFP window (see Note 14).

5. Once each DFRS plasmid-expressing cell is measured, select a region that has no fluorescence and measure the region from each window. This will serve as a background.

6. Copy and paste the Image J results window into an excel worksheet (or similar program) for further analysis.

7. Calculate the corrected total cell fluorescence (CTCF) for both RFP and GFP channels:

   $$\text{CTCF}=\text{Integrated}\text{Density}-(\text{Area}\text{of}\text{selected}\text{cell}\times \text{Mean}\text{of}\text{background}).$$

8. Normalize expression levels by dividing the RFP CTCF by the GFP CTCF.

9. Use the normalized RFP values to produce a graph (Figs. 2b and 3b).

Fig. 2.

Quantifying miR-23a dynamics in single cells during tail regeneration. (a) Control GFP expression (green) and reporter mRFP expression (red) shown at 0, 24, 48, and 96 hour post amputation (hpa). Arrowheads indicate examples of cells that are expressing the DFRS plasmid and are used for quantification. Scale bar is 200 μm. (b) Graphical representation of mRFP expression modulated by miR-23a in the spinal cord during regeneration. mRFP expression is dynamic during the regenerative process, revealing that miR-23a expression is increased at 24 and 96 hpa, while miR-23a levels at 48 hpa are similar to steady state levels of miR-23a (***P < 0.001, NS not significant)