Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.
Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (˜100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.
Part 1A. Equipment
Microscope: upright stereo-dissecting scope with low power objective
L-shaped electrodes:
Electroporation chamber
Part 1B. Reagents
DNA or charged macromolecules
DNA preparation:
* We have had success with vectors containing a strong CMV promoter, such as pCS2+ [16]. For lineage analysis, we usually include DNA encoding green fluorescent protein (pCS2+GFP) at a final concentration of 0.1 μg/μl. [DNA concentrations between 0.1-3 μg/μl were also tested. We found that concentrations below 0.8 μg/μl inefficiently labelled cells, whereas DNA concentrations greater than 2 μg/μl did not improve electroporation efficiency.]
Morpholino oligonucleotide preparation:
(Note: MOs need to be fluoresceinated (3'-carboxyfluorescein modified) or otherwise charged.)
* 0.1-1mM MO solutions were tested. 0.5 mM MO solutions were sufficient for electroporation of many mesenchymal cells.
Micropipettes
Media
2. Electroporation
Micropipette setup
Tadpole preparation
Electroporation
3. Representative Results:
The use of fluorescent molecules allows easy screening of electroporated embryos. Figure 4 shows a typical batch of MO electroporated tadpoles ∼12, 48 and 96 hours after electroporation, incubated at 14.5°C. Using fluorescence microscopy, MOs can be visualised immediately after electroporation and persist for several days after electroporation. In our experience, fluorescence is weakly evident at stage 46 (∼5 days later). In the cartilages, fluorescence decreases dramatically after the onset of differentiation (∼st 42); however, MO fluorescence persists more strongly in other cell types such as the pharyngeal endoderm. Fluorescence microscopy shows that oligonucleotides are incorporated into several craniofacial tissues including cartilage. Oligonucleotide fluorescence can often be visualised in tissue on either side of the head. This is likely due to rapid diffusion of the injection solution throughout the loose craniofacial mesenchyme prior to electroporation.
Figure 1 Homemade electrodes. L-shaped tungsten wire is attached to a 1 ml syringe using non-toxic clay or putty. (A) The electrode terminus measures 5 mm. (B) Attach a pair of electrodes, such that the termini run parallel. Electrodes are attached to pulse generator by DC cables.
Figure 2 Electroporation chamber. (A) 90 mm dish lined with plasticine is filled with media and a T-shaped chamber carved with No 5 watchmaker's forceps. (B) The long side measures 2 mm X 2 mm X10 mm whilst the short measures 2 mm X 2 mm X 5 mm. The head of the embryo rests in the T-junction, ventral side up.
Figure 3 Schematic illustrating electroporation procedure. St. 28 tadpole is placed in electroporation chamber, ventral side up. Micropipette is inserted into facial mesenchyme underlying cement gland. Inject. Micropipette is removed and L-shaped electrodes are aligned parallel flanking the head. Apply eight 50 ms, 20 V square pulses. Retract electrodes. Grow tadpoles to desired stages. Visualise MOs or GFP expression using fluorescence microscopy.
Figure 4 Representative tadpoles 12 (A), 48 (B), and 96 (C) hours post electroporation (stages 30, 34 and 44 respectively). (A"-B") Fluorescent MO can be visualised within craniofacial mesenchyme at stages 30 and 34. Fluorescence can be detected in cartilages at stage 44 (arrowhead, C'-C"). The gut is highly autofluorescent.
In this video, we have demonstrated the feasibility of electroporation-mediated gene delivery into the facial mesenchyme of Xenopus tadpoles. Using this approach, we can bypass early developmental effects of manipulating gene function allowing us to target specific tissues at later time points. Our studies show that heterogenous populations of craniofacial mesenchymal cells can be affected, allowing us to examine lineage of electroporated cells as well as cell autonomous requirements for proteins of interest. Combined with live imaging, we can use this approach to study gene function, over time, during craniofacial development. This novel method highlights the tractability of Xenopus for the study of organogenesis. We anticipate that this method can be broadly adapted to study morphogenesis and differentiation of other tissues as well.
The authors have no conflict of interests.
We are grateful to Nancy Papalopulu and Boyan Bonev for assistance with Xenopus electroporation. We also thank Marc Dionne for critical reading, Jeremy Green and John Wallingford for helpful discussions and members of the Liu lab for their support. This work was funded by grants from the BBSRC (BB/E013872/1) and the Wellcome Trust (081880/Z/06/Z) to KJL.
