Expression and Misexpression of the miR-183 Family in the Developing Hearing Organ of the Chicken

Abstract

The miR-183 family consists of 3 related microRNAs (miR-183, miR-96, miR-182) that are required to complete maturation of primary sensory cells in the mammalian inner ear. Because the level of these microRNAs is not uniform across hair cell subtypes in the murine cochlea, the question arises as to whether hair cell phenotypes are influenced by microRNA expression levels. To address this, we used the chicken embryo to study expression and misexpression of this gene family. By in situ hybridization, expression of all 3 microRNAs is robust in immature hair cells of both auditory and vestibular organs and is present in the statoacoustic ganglion. The auditory organ, called the basilar papilla, shows a weak radial gradient (highest on the neural side) in prosensory cells near the base on embryonic day 7. About nine days later, the basilar papilla also displays a longitudinal gradient (highest in apical hair cells) for the 3 microRNAs. Tol2-mediated gene delivery was used to ask whether cell phenotypes are malleable when the prosensory epithelium was forced to overexpress the miR-183 family. The expression plasmid included EGFP as a reporter located upstream of an intron carrying the microRNA genes. The vectors were electroporated into the otic cup/vesicle, resulting in strong co-expression of EGFP and the miR-183 family that persisted for at least 2 weeks. This manipulation did not generate ectopic hair cells in non-sensory territories of the cochlear duct, although within the basilar papilla, hair cells were over-represented relative to supporting cells. There was no evidence for a change in hair cell phenotypes, such as short-to-tall, or basal-to-apical hair cell features. Therefore, while increasing expression of the miR-183 family was sufficient to influence cell lineage decisions, it did not redirect the differentiation of hair cells towards alternative radial or longitudinal phenotypes.

MicroRNA-183 Family in Inner Ear: Hair Cell Development and Deafness

“…However transfection had no effect on phenotype. No detectable change in hair cell or bundle morphology was seen due to increasd expression by miR-183 family vector [40]. …”

Tol2-Mediated Delivery of miRNAs to the Chicken Otocyst Using Plasmid Electroporation

“…Use the LR Clonase™ II enzyme mix (Invitrogen, Life Technologies) according to the manufacturer's protocol to transfer the miRNA-intron into the pTol2-GFP-Gateway vector [ 12 ]. This will create the desired miRNA-reporter expression vector, now called pTol2-GFP-sd-miR(X)-sa (substitute miR(X) with the name of your miRNA, e.g., pTol2-GFP-sdmiR9-sa).…”

“…Visualize GFP by immunofl uorescence and miR-9 by in situ hybridization. Our lab has successfully collected transgeneexpressing basilar papillas 14 days after electroporation, although survival rates decrease dramatically beyond 3-4 days post-electroporation [ 12 ].…”

“…Most pre-miRNA stem-loop sequences can be found using miRBASE ( http://mirbase.org ; [ 32 ]. It is also possible to construct an intron that carries multiple pre-miR sequences in series [ 12 ]. See manufacturer for further details of Gateway technology (Invitrogen, Life Technologies).…”

“…Even when moderately high viral titers were achieved, subsequent infection of chicken otocysts with RCAS vectors carrying a pre-miRNA intron consistently failed to produce mature miRNAs detectable by in situ hybridization methods. In contrast, inserting identical pre-miRNA-containing introns into plasmid expression vectors, and transducing these into the otocyst via electroporation, were successful [ 12 ];unpublished data).…”