Transfection of mouse cochlear explants by electroporation

Abstract

The sensory epithelium of the mammalian inner ear, also referred to as the organ of Corti, is a remarkable structure comprised of highly ordered rows of mechanosensory hair cells and non-sensory supporting cells located within the coiled cochlea. This unit describes an in vitro explant culture technique that can be coupled with gene transfer via electroporation to study the effects of altering gene expression during development of the organ of Corti. While the protocol is largely focused on embryonic cochlea, the same basic protocol can be used on cochleae from mice as old as P5.

Figure 1

Isolation of developing bony labyrinth of the inner ear. A. Dorsal view of the head of a mouse at E14.5. Dotted line indicates dorsal midline. The skull should be opened along this line followed by removal of the brain. B. Once the brain has been removed, the developing bony labyrinth of the inner ear (outlined) can be visualized in the temporal bone located in the ventral floor of the skull (arrow). The bony labyrinth can be isolated by dissecting around its borders (indicated by arrowheads). C. Ventral view of the isolated bony labyrinths. Cochlear (C) and vestibular (V) regions are indicated. D. Anterior view of the bony labyrinths oriented as in C, illustrating the natural curvature between the cochlear (C) and vestibular (V) regions.

Figure 2

Isolation of the developing cochlear duct and sensory epithelium. A. The bony labyrinth should be oriented with the ventral side up and immobilized by placing two minutien pins through the vestibular region. Once immobilized, it will be possible to identify the base of the cochlear duct through the bony labyrinth. The line drawing on the right illustrates the shape of the cochlear spiral. B. Beginning in the base, use fine forceps to make an opening (straight arrow) and extend it parallel to the duct (curved arrow). C. Use forceps to continue to increase the size of the opening in the bony labyrinth by working along the outside edge of the cochlea (arrow). D. Once the ventral surface of the bony labyrinth of the cochlea has been removed, the developing cochlear duct can be visualized (arrows). E. To expose the developing sensory epithelium of the cochlea, carefully remove the upper (ventral) half of the duct using fine forceps (arrow). Following removal of the upper half of the duct, the remainder of the bony labyrinth of the cochlea can also be removed. F. At this point the developing sensory epithelium (organ of Corti) is completely exposed (arrow). Next, separate the cochlea from the vestibular region of the ear by using fine forceps to cut along the dotted line. G. Ventral view of the isolated cochlear spiral with basal and apical ends indicated. H. In a side view the epithelium is present as a spiral that extends from the base to the apex (arrows). The lower region of the cochlea is comprised of mesenchymal derivatives (note small blood islands, arrowheads) and developing spiral ganglion neurons. I. For electroporation, the cochlea should be oriented between the electrodes with the base located closer to the negative electrode (indicated in the image). J. Schematic diagram depicting the main dissection steps: isolation of the inner ear, removal of overlying cartilage, opening the cochlear duct, and isolation of the cochlear sensory epithelium.

Figure 3

Examples of cochlear electroporations. A. Low magnification image of an explant established on E13.5 and maintained for 6 days in vitro. Hair cells are labeled with an antibody against Myosin6 (red) while transfected cells are labeled with anti-GFP (green). Apex and base of the cochlea are indicated. B. A higher magnification view of a transfected explant treated as in A. Transfected cells are present in Kolliker’s organ (KO), the sensory epithelium (SE) and in the lesser epithelial ridge (LER). C. High magnification view of cells transfected with an Atoh1 expression vector (green). Endogenous hair cells within the SE are labeled with anti-Myosin6 (red). Atoh1-transfected cells located in the SE (arrow) or in KO (arrowheads) have also developed as hair cells. Note that virtually all transfected cells appear yellow as a result of expression of the hair cell marker Myosin6. D. High magnification view of Atoh1-transfected cells within the sensory epithelium. Each transfected cell has developed as a hair cell. E. Cluster of Atoh1-transfected cells located in KO. Actin is labeled in blue. The induction of a group of hair cells leads to an accumulation of actin that is similar to what is observed in the SE. F. Cells transfected with the inhibitory bHLH, Id3 (green), are predominantly inhibited from developing as hair cells.