We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.
Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.
The mammalian organ of Corti is comprised of a mosaic of specialized cell types, including two types of mechanosensory hair cells as well as at least four types of nonsensory supporting cells making it an ideal model system to study normal cellular processes like proliferation, fate specification, differentiation and patterning. In addition, the normal development of these different cell types is essential for normal hearing function. Hence, it is crucial to understand the factors, both molecular and cellular, that regulate their development. However, the small size of the mouse cochlea as well as its inaccessibility poses a particular challenge for gene expression studies. Moreover, most of the cell fate specification and patterning events occur during embryonic time periods and are mostly completed before birth. Therefore, identification and characterization of signaling events during embryonic time periods is essential to gain insight into the molecular basis of cochlear morphogenesis.
Here, we demonstrate a method to culture intact cochlea in vitro from embryonic mouse inner ears. The rationale behind the use of this technique is that cultured cochleae maintain their molecular and morphological characteristics thereby providing a valuable model for investigating potential candidate genes and exploring the mechanisms involved in cochlear morphogenesis. Although transgenic mice can be used for gene expression studies, an in vitro system is often needed for monitoring specific gene functions. Moreover, cochlear cultures can be established from transgenic mouse embryos so that their in vitro development and response to various soluble factors and antagonists can be studied. Although embryos at day 13 (E13) are used in this protocol, cultures from E12 or E14 to early postnatal inner ears can give similar results.
We also present a gene transfer technique in cultured embryonic cochlear explants using square wave electroporation. Following the isolation of the cochlear explants, electroporation can be used to express DNA plasmids of gene(s) of interest in individual cells within the cochlear duct. This technique serves as a complementary approach to studies utilizing transgenic mice to gain insight into the molecular pathways underlying cellular phenotype. Using this method of gene transfer, a variety of epithelial cell types within the embryonic cochlea are transfected, thereby enabling loss-and gain-of-function analyses at the single-cell level. In addition, electrophysiological studies can also be performed in cochlear explant cultures8. This method of in vitro electroporation is relatively simple and straightforward, combined with minimal damage to the tissue, has resulted in a rapid expansion of this technique.
NOTE: All protocols using live animals must be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved methods for the care and use of the laboratory animals. All the dissections should be performed using sterile technique on a clean laminar flow bench. Gloves and a mask, if desired, should be worn during this procedure.
1. Dissection of the Embryonic Mouse Inner Ear
2. Generation of Organ of Corti Explant Cultures
NOTE: At this stage of development, the tissue is cartilaginous and can be easily dissected using forceps.
3. Electroporation-mediated Gene Transfer
4. Analysis of Cochlear Explant Cultures
NOTE: The cultures are usually incubated for 6 DIV which is equivalent to developmental stage, P0. After incubation in vitro, the cultures are fixed and processed for immunocytochemistry.
We describe a method to isolate cochlea from embryonic inner ears and micro-dissect to expose the sensory epithelium. Once dissected, it may be plated and cultured as an intact cochlear duct (Figure 3) and analyzed by immunohistochemistry. The cultured cochlear explants provide a useful assay to examine the effect of variety of soluble factors and pharmacological drugs on cochlear development. Following dissection of cochlear explants, electroporation technique can be used to misexpress genes of interest and examine their effect on cochlear morphogenesis. The results presented here demonstrate that forced expression of basic helix-loop-helix (bHLH) transcription factor, Atoh1 into cultured cochlear explants established from E13 inner ears leads to ectopic hair cell formation. In contrast, forced expression of another bHLH transcription factor, NeuroD1 in prosensory cells and nonsensory epithelial cells within GER leads to the formation of ectopic neurons.
Figure 1: Dissection of the embryonic cochlea. Dissection steps involved in isolating developing cochleae from E13 mouse head as discussed in Part 1. Dotted line in (A) indicates dorsal midline and the borders of inner ears are indicated by arrows in (D). In (E), C indicates cochlea and V, vestibule. Scale bar: 1.0 mm.
Figure 2: Generation of embryonic cochlear explant cultures. Panels (A-F) illustrate key steps in the dissection of the cochlea down to the sensory epithelium as described in Part 2. C, cochlea; V, vestibule. Scale bar: A-E, 100 m; F, 10 mm.
Figure 3: The development of the organ of Corti in cochlear cultures. The formation of hair cells and supporting cells occurs normally in cultured explants. Cross-sectional view of E13 cochlear explant incubated for 6 DIV (equivalent to P0) (B) and top-down view of a whole-mount cochlear explant immuno-stained with Myo7a in green and Sox2 in red demonstrates the presence of one inner and 3-4 rows of outer hair cells and surrounding support cells. Arrows point to Deiters’ cells and black arrowheads indicate the presence of stereociliary bundles within the cochlear explant. Arrowhead in red indicates the depression formed by the prominent pillar cell projection. IHC, inner hair cell; O1,2,3, three outer hair cells, DCs, Deiters’ cells; IPhC, inner phalangeal cell; GER, greater epithelial ridge which includes nonsensory epithelial cells present on the modiolar side of the epithelium; LER, lesser epithelial ridge, includes nonsensory epithelial cells present on strial edge of the sensory epithelium. Scale bar: B, 100 µm.
Figure 4: Electroporation of Atoh1.EGFP (A-C) and NeuroD1 into the organ of Corti explant culture. Embryonic (E) day 13 cochlear explants were established from WT CD1 mouse pups and electroporated with Atoh1.EGFP (A-C), NeuroD1-EGFP (D-F) reporter constructs as described and immunolabeled with hair cell-specific marker anti-Myo7a or neuronal marker, TuJ1 (β-tubulin III) in red. Electroporated cells which can be visualized by EGFP expression are seen throughout the GER, LER and sensory epithelium (SE) cells. The NeuroD1 transfected cochlear epithelial cells after 5 DIV acquire neuronal phenotype with dendritic processes, most of which are positive for TuJ1 while Atoh1 transfected cells are positive for Myo7a expression. SE, sensory epithelium; GER, greater epithelial ridge. Scale bar: A-C, 20 µm; D-F, 50 µm. Please click here to view a larger version of this figure.
Name | Company | Catalog # |
HBSS | Gibco | 14065-056 |
HEPES | Gibco | 15630-080 |
Dulbecco’s Modified Medium | Gibco | 12430-054 |
Fetal Bovine Serum | Gibco | 10082 |
N-2 Supplement (100X) | Gibco | 17502-048 |
Ciprofloxacin Hydrochloride | Cellgro | 61-277-RF |
Glass Dish 60mm | Kimble Chase | 23062-6015/23064-6015 |
Glass Dish 100 mm | Kimble Chase | 23064-10015/23062-10015 |
Minutien Pins | Fine Science Tools | 26002-15 |
Dumont # 5 Forceps | Fine Science Tools | 11251-10 |
Pulse Generator | Protech International Inc | CUY21Vivo-SQ |
Glass Bottom Culture Dishes | MatTek | P35G-0-10-C |
Matrigel Matrix | BD Biosciences | 356237 |
Culture Dish, 60 X 15 mm | Becton Dickinson | 353037 |
Tissue Culture Dishes | Greiner Bio-one | 639160 |
Phosphate Buffered Saline | Gibco | 10010-023 |
OS-30 | Dow Corning | 4021768 |
Fluoromount | Southern Biotech | 0100-01 |
Conical Tubes, 15ml | Greiner Bio-one | 188261 |
Myosin 6 | Proteus Biosciences Inc | 25-6791 |
Myosin 7a | Proteus Biosciences Inc | 25-6790 |
TuJ1 | Sigma | T2200 |
Table 1: List of reagents and tools used for establishing cochlear explants and electroporation.
–>All the cells within the membranous labyrinth of the mouse inner ear including sensory, nonsensory and the spiral ganglion neurons are all derived from placodally-derived otocyst located adjacent to the hindbrain of the ectoderm, around E810-14. At E11, the ventral region of the otocyst extends to form the cochlear duct and as development continues, a group of epithelial cells within the cochlea, as well as in other regions of the otocyst, become specified as prosensory patches that will subsequently give rise to different types of mechanosensory hair cells and nonsensory supporting cells. In the developing cochlea, one row of inner hair cells and three rows of outer hair cells can be identified around E15.5 and by E17, patterning is essentially complete with single row of inner hair cells, pillar cells and three rows of outer hair cells. In a period of time that spans from E11 when the cochlear duct first starts to grow out through E17 all of the different cell fate decisions and patterning occur within the developing epithelium to generate a striking cellular pattern with a normal complement of hair cells and support cells. Loss of hair cells and/or support cells is the leading cause of hearing impairment. Since these cell types are generated only during a fairly compact time period during embryonic development, it is crucial to understand the molecular and genetic pathways that specify each of these cell types which should lead to significant insight into regenerative strategies.
Cochlear culturing and electroporation techniques have been developed to manipulate gene expression in the developing mouse. In this video, we have demonstrated culturing techniques for generating primary explants and an electroporation technique for gene delivery into cultured embryonic murine organ of Corti. The primary explants prepared in this fashion can be maintained for 7-10 days in vitro. Cochlear explants can be used to manipulate gene expression by pharmacological approaches which will enable us to understand the mechanisms that regulate developmental processes such as fate specification, commitment, differentiation, and patterning. Moreover, this method facilitates the analysis of developmental phenotypes of mutant embryonic mice that do not survive past E12.
The square wave electroporation-mediated gene transfer procedure provides a mechanism for manipulating gene expression in sensory hair cells, support cells, and cells within GER and LER regions in order to visualize their fate ex vivo. Using this procedure, we can downregulate or ectopically express specific genes in prosensory cells, hair cells and/or supporting cells in an otherwise wild-type background and analyze their specific effects on fate specification and differentiation. This will enable us to understand specific gene function within the developing cochlea. For example, as shown in Figure 4, the forced expression of Atoh12,4 or NeuroD18 in cells within GER or LER leads to the formation of ectopic hair cells and neurons respectively. In addition, this technique allows to electroporate multiple candidate genes6,7 and examine their effect on sensory hair cell and supporting cell formation, differentiation and their organization. The gene transfer technique involving adenoviral vectors offers broad expression and have been successfully used in the inner ear15,16. However, this technique depends on the skill to generate and purify the viruses which is often time-consuming.
Although cochear cultures can be established as early as E12, electroporation of cochlear explants younger than E12.5/E13 will result in damage to the tissue making it cumbersome for analysis. In addition, the promoter of the expression vector used determines which cell types are transfected within the cochlear duct. For example, the human cytomegalovirus promoter containing expression vectors yields robust transfection in Kollikers’ organ, while use of CMV early enhancer/chicken beta actin promoter results in higher efficiency of transfection in cells within the sensory epithelium. The common problems encountered during the electroporation of embryonic cochlear explants are excessive cell death and/or damage of the tissue and poor transfection efficiency. The appropriate spacing of the electrodes and DNA concentration plays a crucial role in obtaining minimal damage and higher transfection efficiency. In summary, these techniques enhances our ability to manipulate gene expression via gain or loss of function strategies and pharmacological manipulations and greatly aid in dissecting signals that influence patterning and cell fate decisions.
The authors have nothing to disclose.
We would like to acknowledge Dr. Bradley Schulte for comments on this protocol. This work was supported by National Institutes of Health grant R00 (5R00DC010220). This project was performed in a renovated laboratory space supported by Grant C06RR014516.
HBSS | Gibco | 14065-056 | |
HEPES | Gibco | 15630-080 | |
Dulbecco’s Modified Medium | Gibco | 12430-054 | |
Fetal Bovine Serum | Gibco | 10082 | |
N-2 Supplement (100X) | Gibco | 17502-048 | |
Ciprofloxacin Hydrochloride | Cellgro | 61-277-RF | |
Glass Dish 60mm | Kimble Chase | 23062-6015/23064-6015 | |
Glass Dish 100 mm | Kimble Chase | 23064-10015/23062-10015 | |
Minutien Pins | Fine Science Tools | 26002-15 | |
Dumont # 5 Forceps | Fine Science Tools | 11251-10 | |
Pulse Generator | Protech International Inc | CUY21Vivo-SQ | |
Glass Bottom Culture Dishes | MatTek | P35G-0-10-C | |
Matrigel Matrix | BD Biosciences | 356237 | |
Culture Dish, 60 X 15 mm | Becton Dickinson | 353037 | |
Tissue Culture Dishes | Greiner Bio-one | 639160 | |
Phosphate Buffered Saline | Gibco | 10010-023 | |
OS-30 | Dow Corning | 4021768 | |
Fluoromount | Southern Biotech | 0100-01 | |
Conical Tubes, 15ml | Greiner Bio-one | 188261 | |
Myosin 6 | Proteus Biosciences Inc | 25-6791 | |
Myosin 7a | Proteus Biosciences Inc | 25-6790 | |
TuJ1 | Sigma | T2200 | |