Analysis and functional evaluation of the hair-cell transcriptome

Abstract

An understanding of the molecular bases of the morphogenesis, organization, and functioning of hair cells requires that the genes expressed in these cells be identified and their functions ascertained. After purifying zebrafish hair cells and detecting mRNAs with oligonucleotide microarrays, we developed a subtractive strategy that identified 1,037 hair cell-expressed genes whose cognate proteins subserve functions including membrane transport, synaptic transmission, transcriptional control, cellular adhesion and signal transduction, and cytoskeletal organization. To assess the validity of the subtracted hair-cell data set, we verified the presence of 11 transcripts in inner-ear tissue. Functional evaluation of two genes from the subtracted data set revealed their importance in hair bundles: zebrafish larvae bearing the seahorse and ift 172 mutations display specific kinociliary defects. Moreover, a search for candidate genes that underlie heritable deafness identified a human ortholog of a zebrafish hair-cell gene whose map location is bracketed by the markers of a deafness locus.

Fig. 1.

Expression of hair-cell transcripts in the anterior macula. The schematic diagram of the left ear of a 4-day-old zebrafish larva depicts the sensory maculae (blue), cristae of the semicircular canals (green), and otoliths (gray). A red box delimits the anterior macula. In situ hybridization was performed on whole-mount embryos for selected genes identified in Table 1. A bracketed symbol for a human or mouse gene indicates a probe designed to hybridize with a zebrafish transcript for which the corresponding protein's amino acid sequence most resembles that encoded by the indicated gene. The rims2 sense probe and the myo6 probe constituted, respectively, a negative and a positive control. (Scale bars, 5 μm.)

Fig. 2.

Kinocilia in wild-type and ift172 mutant zebrafish. (A) In a confocal image of the crista from a semicircular canal in a wild-type animal, immunolabeling of acetylated tubulin shows prominent kinocilia. (B) The corresponding image from an ift172 mutant reveals no kinocilia. For this and subsequent instances in which a receptor organ lacks kinocilia, an asterisk indicates the apical hair-cell surface. (C) Numerous kinocilia are apparent in a control preparation from the anterior macula. (D) Although the outlines of hair cells are clearly visible in the anterior macula of a mutant, all of the kinocilia but one are missing. (E) A neuromast from the lateral line of a wild-type animal displays a cluster of kinocilia. (F) The kinocilia are absent from a mutant. (Scale bars, 5 μm.)

Fig. 3.

Effect of the seahorse mutation on the kinocilia of 4-day-old zebrafish. (A) Numerous kinocilia, labeled by an antiserum against acetylated α-tubulin, protrude from the crista of semicircular canal in a wild-type animal. (B) The kinocilia of a seahorse mutant larva displays enlarged kinocilia, one with a basal swelling. (C) In another mutant animal, several kinocilia are bloated at their bases. (D) A higher-magnification view of one hair cell from a seahorse larva depicts the swollen base of a kinocilium; the arrowhead indicates the position of the hair cell's apical surface. (Scale bars, 5 μm.)