Mice Doubly Deficient in the Midkine and Pleiotrophin Genes Exhibit Deficits in the Expression of B-Tectorin Gene and in Auditory Response
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Laboratory Investigation (2006) 86, 645–653 & 2006 USCAP, Inc All rights reserved 0023-6837/06 $30.00 www.laboratoryinvestigation.org Mice doubly deficient in the midkine and pleiotrophin genes exhibit deficits in the expression of b-tectorin gene and in auditory response Peng Zou1, Hisako Muramatsu1,2, Michihiko Sone3, Hideo Hayashi3, Tsutomu Nakashima3 and Takashi Muramatsu1,4 1Department of Biochemistry, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, Japan; 2Division of Animal Models, Center for Neurological Disease and Cancer, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, Japan; 3Department of Otolaryngology, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya, Japan and 4Department of Health Science, Faculty of Psychological and Physical Sciences, Aichi Gakuin University, Nisshin, Aichi, Japan a-Tectorin and b-tectorin are major noncollagenous proteins of the tectorial membrane, which plays a crucial role in the reception of sonic signals in the cochlea. Midkine and pleiotrophin are closely related proteins that serve as growth factors and cytokines. In mice doubly deficient in the midkine gene and pleiotrophin gene, expression of b-tectorin mRNA was nearly abolished in the cochlea on day 1 and 7 after birth. Expression of a- tectorin mRNA was unaffected in the double knockout mice, and expression of b-tectorin mRNA was not altered in mice deficient in only the midkine or pleiotrophin gene. In newborn wild-type mice, both midkine and pleiotrophin were expressed in the greater epithelial ridge of the cochlea, in which b-tectorin mRNA was strongly expressed. These results indicate that either midkine or pleiotrophin is required for significant expression of b-tectorin. In agreement with the view that b-tectorin is essential for normal auditory function, mice doubly deficient in both midkine and pleiotrophin genes exhibited very severe auditory deficits. We observed that mice deficient in either midkine or pleiotrophin gene were also impaired in their auditory response, but the level of the deficit was generally low or moderate. The present finding illustrates the importance of growth factor expression in the cochlea for auditory function. Laboratory Investigation (2006) 86, 645–653. doi:10.1038/labinvest.3700428; published online 17 April 2006 Keywords: auditory response; cochlea; growth factor; midkine; pleiotrophin; b-tectorin Genetic studies have revealed molecular basis of by the tectorial membrane. b-Tectorin is also deficits in auditory function in certain cases.1–8 abundantly expressed in the tectorial membrane.9 However, the entire picture of abnormalities causing a- and b-Tectorins have protein motifs involved in auditory deficits is not yet provided. So far, a- sperm–egg adhesions; the motifs appear to function tectorin is one of the most studied molecules in in the formation of filament-based matrices or relation to auditory function. Mutation of the a- gels.9,10 Although direct evidence of the participa- tectorin gene leads to auditory deficits in humans,1,2 tion of b-tectorin in auditory function is lacking, its and mice deficient in the a-tectorin gene also exhibit expression is decreased upon thyroid hormone deficits in auditory function.3 a-Tectorin is a major deficiency,11 which causes auditory impairment. protein of the tectorial membrane in the cochlea, in Furthermore, b-tectorin is an antigen that causes which the sound-induced movement of the basilar autoimmune hearing loss.12 Here, we report that membrane is transmitted to the sensory hair bundles mice doubly deficient in midkine (MK) and pleio- trophin (PTN) are impaired in b-tectorin expression and exhibit severe deficits in auditory function. Correspondence: Dr H Muramatsu, PhD, Department of Bio- MK and PTN, also called heparin-binding growth- chemistry, Nagoya University Graduate School of Medicine, associated molecule (HB-GAM), are low molecular 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. weight basic proteins with closely related structures E-mail: [email protected] Received 15 October 2005; revised and accepted 13 March 2006; and serve as growth/differentiation factors and published online 17 April 2006 cytokines.13–19 The expression of these factors is The midkine family and b-tectorin P Zou et al 646 temporally and spatially regulated; intense expres- Expression Bioarray System (Amersham Bio- sion is usually observed in tissues during the sciences) provided by Kurabo (Osaka, Japan). embryonic and perinatal periods.20,21 Overexpres- sion of these factors, especially MK, in adult tissues is associated with malignancy and inflammatory Preparation of Specimens from Cochleas diseases.16,18,22–25 Both MK and PTN are strongly expressed in the Six cochleas from postnatal day (P) 7 or P14 mice central nervous system during development20,21 and were fixed in 4% paraformaldehyde in phosphate- 1 promote the neurite outgrowth, and migration and buffered saline (PBS; pH 7.4) for 1 day at 4 C, survival of neurons.16,18,19,26–28 Furthermore, PTN washed three times with PBS, and decalcified in 1 promotes differentiation of neural progenitor 0.5 M EDTA (pH 7.5) for 7 days at 4 C. After cells.29,30 Mice that are deficient in the MK or PTN dehydration through a graded series of ethanol gene exhibit certain behavioral abnormalities.31,32 solutions, the specimens were embedded in paraf- Overexpression studies also indicate that MK fin. Sections were cut at 5 mm, placed on glass slides promotes neurogenesis in Xenopus and zebra fish coated with 3-aminopropyltriethoxysilane, and then embryos.33,34 In a recent study in zebra fish, which subjected to staining with hematoxylin and eosin have two species of MK due to gene duplication, it (HE), or to in situ hybridization. Total cochlear RNA was established that a form of MK plays essential was isolated from six cochleas of WT, MKKO, roles in the formation of the medial floor plate, one PTNKO or DKO mice using Isogen (Wako, Tokyo, of the most ventral neural tube structures.35 The Japan) according to the manufacturer’s directions. factors are also expressed in specific regions during development of the sensory organs, suggesting roles in the developmental process.20,21 MK and PTN have Reverse Transcription (RT)-PCR 45% sequence identity at the protein level, and 14,16,18,19,36,37 Total cochlear RNA (500 ng) was subjected to RT in a share many of the in vitro activities. 20 ml reaction mixture containing 40 Us of RNase Therefore, we inferred that the functions of MK and inhibitor (Invitrogen, Carlsbad, CA, USA) and 0.5 mg those of PTN frequently overlap. Thus, to clarify the of oligo dT primers (Invitrogen). In all, 50 U of roles of these factors in vivo, we generated mice SuperScriptt II reverse transcriptase (Invitrogen) doubly deficient in both the MK and PTN genes. As was used to synthesize double-stranded cDNA. The a result of many in vitro activities of these factors to 50-ml PCR mixture contained 2 ml of the product of neuronal cells, we focused to the possible abnorm- the RT, 5 mlof10Â PCR buffer, 2 mM dNTP, 2.5 U of alities in the nervous and sensory systems of the ExTaq DNA polymerase (Takara, Kyoto, Japan), and doubly deficient mice. Consequently, we were able to 20 pmol each of the primers. After preincubation at demonstrate that expression of growth factors in the 941C for 5 min, PCR was performed for 30 cycles of cochlea is important in normal auditory function. 30 s at 941C, 40 s at 601C, and 90 s at 721C, followed by one extension-cycle for 5 min at 721C. The oligonucleotides used for amplification were as Materials and methods follows: MK (50-ATGCAGCACCGAGGCTTCTT-30 0 0 0 Knockout Mice and 5 -CTTTGACTTGGTCTTGGAGG-3 ); PTN (5 - ACTGGCGCCGAGTGCAAACAG-30 and 50-GAGCTT Mice deficient in the MK gene (Mdk)24 were GCCACAGGGCTTGGA-30); a-tectorin (50-AGCAAC generated as described previously. Mice deficient GACAAGCTTCGGTACTTC-30 and 50-TCTTGCAGG in the PTN gene (Ptn) were also described38 GATCCGGTCACACAG-30); b-tectorin (50-GAAATCA (Muramatsu et al, submitted for publication). After TGCACTCCGAATAAAGC-30 and 50-CGAGGGGGTG the backcrossing of Mdk þ /À or Ptn þ /À to C57BL/6J GCCCAGCAGCTATT-30); b-actin (50-GTGGGCCGCT mice for 10 generations, Mdk þ /À and Ptn þ /À mice CTAGGCACCA-30 and 50-CGGTTGGCCTTAGGGTT were mated with each other to generate wild-type CAGGGGG-30). (WT), MK knockout (MKKO) PTN knockout (PTNKO) and double knockout (DKO) mice, which were used in the present study. The female sterility Real-Time Quantitative PCR of DKO will be described elsewhere (Muramatsu H et al, submitted for publication). Real-time quantitative PCR for a-tectorin, b-tectorin and b-actin was carried out using the TaqMan probe and TaqMan Universal PCR Master Mix according to DNA Microarray Analysis the manufacture’s directions. Expression of each gene was normalized to the amount of b-actin RNA. At day 14 after birth, total RNA from the whole brain We calculated relative amounts of each product was isolated with an RNeasy column (QIAGEN using the comparative CT method as described in Sciences, Tokyo, Japan) according to the manufac- the user bulletin of ABI Prism 7500 Sequence turer’s protocol. The gene expression profile was Detection System (Applied Biosystems, Foster City, analyzed with a DNA microarray using a CodeLink CA, USA). Laboratory Investigation (2006) 86, 645–653 The midkine family and b-tectorin P Zou et al 647 In Situ Hybridization PTNKO and DKO groups. After the mice were anesthetized using a mixture of ketamine hydro- MK cDNA (nucleotides 1-420)39 or PTN cDNA 40 chloride, xylazine, and acepromazine maleate ad- (nucleotides 270-773) was inserted into Bluescript ministered subcutaneously, needle electrodes were KSII þ (Stratagene). The PCR product of a-tectorin inserted into the vertex (positive), occiput (nega- (469 bp) or b-tectorin (576 bp) was also subcloned tive), and hindpaw (ground).