Signatures from tissue-specific MPSS libraries identify transcripts preferentially expressed in the mouse inner ear - PubMed (original) (raw)

Signatures from tissue-specific MPSS libraries identify transcripts preferentially expressed in the mouse inner ear

Linda M Peters et al. Genomics. 2007 Feb.

Abstract

Specialization in cell function and morphology is influenced by the differential expression of mRNAs, many of which are expressed at low abundance and restricted to certain cell types. Detecting such transcripts in cDNA libraries may require sequencing millions of clones. Massively parallel signature sequencing (MPSS) is well suited to identifying transcripts that are expressed in discrete cell types and in low abundance. We have made MPSS libraries from microdissections of three inner ear tissues. By comparing these MPSS libraries to those of 87 other tissues included in the Mouse Reference Transcriptome online resource, we have identified genes that are highly enriched in, or specific to, the inner ear. We show by RT-PCR and in situ hybridization that signatures unique to the inner ear libraries identify transcripts with highly specific cell-type localizations. These transcripts serve to illustrate the utility of a resource that is available to the research community. Utilization of these resources will increase the number of known transcription units and expand our knowledge of the tissue-specific regulation of the transcriptome.

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Figures

Figure 1

Schematic representation of tissues dissected for RNA isolation in the production of the inner ear MPSS libraries. The MoVB library was made from microdissected sensory epithelia of the vestibular receptor organs. Red shading indicates the approximate locations of sensory epithelia patches comprising utricular and saccular maculae and three cristae ampulares. The right panel shows a cross section through one turn of the cochlea. The approximate boundaries of the dissected material used to generate the organ of Corti (MoCR) and stria vascularis (MoSV) libraries are indicated. CC Claudius cells; DC, Deiter’s cells; ESC, external sulcus cells; HC, Hensen cells; IDC, interdental cells; IHC, inner hair cell; IPC, inner pillar cell; ISC, inner sulcus cells; OHC, outer hair cells; OPC, outer pillar cells; RM, Reissner’s membrane; SP, spiral prominence; SV, stria vascularis. The cross-section of the cochlea is redrawn from the Hereditary Hearing Loss Homepage (

http://webhost.ua.ac.be/hhh/

), with the permission of Guy Van Camp.

Figure 2

RT-PCR experiments confirming expression of predicted transcripts in the inner ear. Templates were mRNA derived from liver, kidney, pancreas, retina, brain, testes, inner ear and genomic DNA. Primers were designed to span at least one intron at the 3′ end of a transcript predicted by an MPSS signature. Control primers for the mouse beta-actin gene, Actb, amplify different size products from cDNA and genomic DNA templates, and are used in RT-PCR as a loading control. The RT-PCR products depicted here were cloned and sequenced, and were the templates for in situ RNA probes.

Figure 3

In situ hybridization in cross sections of the mouse inner ear. A) Antisense probe for Vmo1 shows discrete localization of the signal to Reissner’s membrane in cross sections through several cochlear turns. B) Control (sense) probe for Vmo1. C) A similar section to A and B shows hybridization signals obtained using an antisense probe for GM414, a mouse homolog of the zebrafish gene Otolin-1. D) The same probe as in C shows dense signal for GM414 in the maculae of the utricle and saccule. E) Control (sense) probe results for GM414 in the utricle (u) and saccule (s) in a section adjacent to the one depicted in panel D. F) A cross section through one turn of the cochlea at approximately the same level as the boxed region in panel B. Discrete localization of the Scg2 message to cells of the spiral prominence. G) An antisense probe for Umodl1 (olfactorin) reveals discrete localization of the message restricted to the middle of the organ of Corti, possibly in the hair cells or their supporting cells. H) An antisense probe for A430025D11Rik shows hybridization signal in the organ of Corti despite the lack of any signature in the MoCR library. A signature for this gene is found at moderate levels (160 tpm) in the MoVB library. In situ hybridization confirms expression in the vestibular sensory epithelia (data not shown). I) Cib3 in situ hybridization signal in a lateral crista ampularis (lc) and the macula of the utricle (mu). The intense signal on the slopes of the crista and faint signal at the apex is consistent with either supporting cells or hair cells. J) widespread in situ hybridization signal for antisense probe to the novel gene 1110017I16Rik. Scale bars = 100 μm.

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Signatures from tissue-specific MPSS libraries identify transcripts preferentially expressed in the mouse inner ear - PubMed (original) (raw)