Cell type-specific markers offer advantages that range from identification of cells by immunolabeling, ability to cell sort and enrich a particular cell type from a heterogenous population, to functional studies of these proteins using mouse models. Specifically, due to their wide use in FACS, there is a large variety of commercially available fluorochrome-conjugated antibodies against CD proteins that can be used for cell sorting or cell tracking in vivo (Beare et al. 2008
). Using a combination of microarray data analysis, comparisons of the expression profiles of auditory and vestibular sensory epithelia from newborn mice, followed by validation with real-time RT-PCR, we have established the expression of 68 CD genes in the newborn mouse inner ear. We focused our study on ears of early postnatal mice. By P0, most of the cell types of the mouse inner ear are present; however, the sensory epithelia are yet to be fully differentiated (reviewed in Kelley 2007
). Our research focus was to identify novel cochlea-specific markers. In addition, our semi-quantitative real-time RT-PCR assays addressed differential patterns of gene expression in the discrete vestibular patches (utricle, saccule, and cristae). This study is unique in its attempt to differentially quantify a large set of genes in the distinct vestibular patches using semi-quantitative real-time RT-PCR.
Our analysis identified two markers that were uniquely enriched in the cochlear sensory epithelium, CD44 and Fgfr3. Interestingly, the real-time RT-PCR analysis revealed several patterns of vestibular epithelia gene expression. A small subset of genes were specifically enriched both in the cochlear and saccular sensory epithelia. These included CD10, CD26, CD56, CD83, CD91, CD106, CD140a, CD140b, CD248, and CD302. In contrast, none of the genes were uniquely enriched either in the cochlear and utricular epithelia or in the cochlear epithelium and the epithelia of the cristae ampullaris. Indeed, a very small number of markers were specifically enriched in the epithelia of the cristae ampullaris. Further localization of these proteins by immunohistochemistry will shed light on the functional significance of these results.
While hair cells have multiple cell type-specific markers that are expressed from early developmental stages and persist to adulthood, e.g., myosin VI (Hasson et al. 1997
), myosin VIIa (Hasson et al. 1995
), Pou4f3 (Xiang et al. 1998
), and prestin (Belyantseva et al. 2000
), similar cell type-specific markers for supporting cells have not been identified. Moreover, while the supporting cells differ in structure and function, most of the supporting cell-specific markers are shared by at least two cell types. For example, SOX2, a transcription factor, labels six different support cell types within the cochlea including pillar cells, while a different transcription factor, PROX1, labels pillar cells and Deiters’ cells (Hume et al. 2007
). Previous studies have also identified FGFR3, a cell surface marker, as expressed by developing pillar and Deiters’ cells (Mueller et al. 2002
We have identified CD44 as the first outer pillar cell-specific marker in the early postnatal mouse inner ear. Like many other supporting cell markers, the expression of CD44 is not limited to the outer pillar cells. However, the lack of CD44 staining in other cells immediately adjacent to the outer pillar cells within the sensory epithelium, makes it an ideal marker for the outer pillar cells in the early postnatal period. CD44 is an integral cell membrane glycoprotein with a diverse range of suggested functions (MIM 107269). The principal ligand of CD44 is hyaluronic acid, an extracellular matrix protein (Aruffo et al. 1990
). CD44 has been primarily studied as a receptor expressed on activated T cells and is known as a lymphocyte homing receptor (Aruffo et al. 1990
; Stefanova et al. 1989
). By binding hyaluronic acid, CD44 can facilitate extravasation of lymphocytes at sites of inflammation. Other identified ligands include laminin, fibronectin, collagens, serglycin, and osteoponin (reviewed in Goodison et al. 1999
). CD44 consists of extracellular, transmembrane, and intracellular domains. The mouse CD44
consists of nine constant exons that flank ten variable exons. The transmembrane and intracellular domains of CD44 are encoded by the constant exons, while the alternatively spliced exons affect the structure of the extracellular domain (Goodison et al. 1999
). Specific splice variants of the protein have since been implicated in malignant transformation, cancer metastasis, and inflammatory diseases (reviewed in Bourguignon 2008
; Johnson and Ruffell 2009
; Liu and Jiang 2006
). The expression of CD44 has also been described in multiple morphogenetically active epithelia as well as in Muller cell apical microvili in the retina (Stefanova et al. 1989
; Yu and Toole 1997
). Using in situ hybridization, CD44
v4-7, v4-5, v6-7, v8-10, and the short isoform were detected in E15 inner ears (Yu and Toole 1997
). Our results show that by P0, the expression of the short isoform of CD44
as well as CD44 v8-10
persists. CD44 v6-10
could also be detected. Other isoforms were not found, and it is possible that they are expressed only at earlier developmental stages.
KO mice are viable and do not display obvious developmental defects, but do suffer from specific alterations in their lymphocyte-dependent immune responses (Protin et al. 1999
; Schmits et al. 1997
). Our results show that in the absence of CD44, the mouse inner ear develops normally and that ABR hearing thresholds are comparable to wild-type and heterozygous littermates. In addition, specific blocking experiments of cochlear explant cultures harvested at E14.5 and incubated with a blocking antibody for CD44 did not result in any discernable phenotype until a toxic dose was reached (data not shown). Therefore, the specific role of CD44 and its isoforms in the mouse inner ear remains to be determined. Furthermore, as we could not identify mutations in CD44
among the human DFNB51 families, mutations of CD44
appear not to have a role in human hereditary hearing loss.
Finally, the mammalian auditory and vestibular sensory epithelia are highly complex and composed of a variety of tightly organized cell types. Recently, mice with transgenic expression of a green fluorescent protein (GFP) under the regulation of general supporting cell-specific (p27kip1
) or hair cell-specific (Atoh1
) promoters were used to isolate these mixed cell populations, respectively (Doetzlhofer et al. 2006
; White et al. 2006
). Similarly, mice with cell type-specific GFP expression have been used to isolate and characterize the transcriptomes of specific cell populations from the brain and the retina (Ivanov et al. 2008
; Lobo et al. 2006
; Marsh et al. 2008
). Antibodies to CD proteins are routinely used for FACS analysis of leukocytes, routine diagnostics of hematologic diseases, and for staining pathology slides for cancer diagnostics. Our results indicate that multiple CD genes are expressed in the mouse auditory and vestibular sensory epithelia, and suggest that at least some of these proteins may have differential expression patterns in the inner ear, similar to CD44. By further characterizing the expression pattern of other CD genes in the mouse inner ear, it may be possible to devise protocols for cell type-specific sorting from wild-type mice. This would allow for comparative analysis of inner ear cell type-specific expression profiles of wild-type and mutant mice and could assist in the identification of new deafness genes.