Introduction

MicroRNAs (miRNAs) are 20–25 nucleotide long noncoding RNAs that have been found in a wide variety of organisms and shown to exert essential roles by regulating the stability and translation of target messenger RNAs (mRNAs) (1,2,3,4). Interestingly, most miRNAs show tissue-specific and developmentally regulated expression (5,6,7,8). To investigate the role played by miRNAs during development, the establishment of techniques allowing the detection/monitoring of miRNA expression during cell fate change in vivo is crucial (9). To detect miRNAs in tissues microscopically, two ingenious approaches have been used: (i) in situ hybridization using locked nucleic acid (LNA)-modified DNA oligonucleotide probes, which detect the presence of miRNAs irrespective of their potential activity (8) and (ii) the expression or administration of target mRNAs (sensors), which detect miRNAs via their degradation-triggering activity toward the sensor (10,11,12).

Although both approaches are powerful, certain limitations remain. Thus, in situ hybridization using LNA probes requires tissue fixation, which prevents the monitoring of miRNA appearance/disappearance in a given cell lineage during cell fate change. While this limitation could potentially be overcome by in vivo expression of a sensor mRNA encoding a fluorescent protein, the latter approach has typically involved the generation of transgenic animals (10,11). Moreover, in the sensor approach, a lack of signal is interpreted as being indicative of the presence of a miRNA, which calls for some means of verification that the sensor mRNA is actually being transcribed in the cell lacking sensor protein. Overcoming these limitations, we report here a relatively simple and reliable system that allows the detection of miRNAs with cellular resolution in vivo without the need to generate transgenic animals.

Materials and Methods

DFRS Plasmids

Details concerning the construction of the dual-fluorescent green fluorescent protein (GFP)-reporter/monomeric red fluorescent protein (mRFP)-sensor (DFRS) plasmids used are provided in the supplementary materials available online at www.BioTechniques.com.

Zebrafish Embryo Injection

All embryos were obtained from the zebrafish AB wild-type line. Single blastomeres of 2- to 8-cell stage embryos were injected with approximately 500 pL phosphate-buffered saline (PBS) containing, as the standard concentration, 0.1 g/L purified DFRS plasmid. In some experiments, a higher concentration (0.5 g/L) was used. Embryos were maintained in E3 medium and manipulated by standard methods (13).

Mouse Embryo Electroporation

In utero electroporation of mouse embryos was performed as described (14), except that the topology of the embryos was determined using illumination and a dissecting microscope rather than ultrasound microscopy. Pregnant mice 13 days postcoitum were anesthetized with isofluorane vapor and their uteri exposed. Using a glass capillary, 1–3 L PBS containing 3–5 g/L DFRS plasmid were injected through the uterine wall into the lumen of the telencephalic vesicles or released in proximity of the ectoderm of the embryo. Immediately after injection, 6 square electrical pulses of 30 V, 50 ms each at 1-s intervals were delivered through platinum electrodes (2 mm diameter) using a BTX®-ECM®830 electroporator (Harvard Apparatus, Holliston, MA, USA). The orientation of the electric field was used to direct the uptake of the plasmid to specific regions of the developing brain or ectoderm. After electroporation, the uterus was relocated into the peritoneal cavity, and the abdomen was sutured. Mice were sacrificed either 24 or 72 h after in utero electroporation, and the embryos were collected for further analyses. Ex utero electroporation of DFRS plasmids into telencephalic vesicles of E10 mouse embryos followed by 24 h of whole-embryo culture was performed as described previously (15).

In Situ Hybridization on Cryosections Using LNA-Modified Oligonucleotides

Whole-mount E11 and E14 mouse embryos were fixed overnight at 4°C in 4% paraformaldehyde in 120 mM phosphate buffer, pH 7.4, equilibrated in 30% sucrose in PBS, and embedded in Tissue-Tek®, and 10-m cryosections were prepared. In situ hybridization was performed according to standard protocols with the following modifications. LNA-modified DNA oligonucleotides (Exiqon A/S, Vedbaek, Denmark) were labeled with digoxygenin (DIG)-ddUTP using the DIG oligonucleotide 3′ end labeling kit (Roche Diagnostic GmbH, Mannheim, Germany) according to manufacturer's instructions. Prehybridized cryosections were incubated overnight at 51°C (miR-9) or 59°C (miR-124a) in hybridization buffer containing 200 pmol/mL of DIG-labeled LNA-oligonucleotide. Following incubation with alkaline phosphatase-conjugated anti-DIG antibody at 4°C overnight, staining with 5-bromo-4-chloro-3-indoxyl phosphate/nitro blue tetrazolium (BCIP/NBT; Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) was done at 37°C for 2 h and then either at 4°C for 1–2 days or at room temperature for 6–12 h. Images were acquired with a standard upright microscope (Olympus® Optical, Europe GmbH, Hamburg, Germany).