Loss of liver kinase B1 causes planar polarity defects in cochlear hair cells in mice

doi:10.1007/s11684-016-0494-3

Front. Med.

2016, Vol. 10

Issue (4) : 481-489 https://doi.org/10.1007/s11684-016-0494-3

RESEARCH ARTICLE

Loss of liver kinase B1 causes planar polarity defects in cochlear hair cells in mice

Yuqin Men¹,Aizhen Zhang¹,Liwen Zhang²,Yecheng Jin¹,Zhishuo Wang¹,Jing Zhao¹,Xiaolin Yu¹,Jian Zhang¹(

),Jiangang Gao¹(

)

¹. School of Life Science and Key Laboratory of the Ministry of Education for Experimental Teratology, Shandong University, Jinan 250100, China
². Jinan First People’s Hospital, Jinan 250011, China

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Abstract

The tumor suppressor gene liver kinase B1 (LKB1), also called STK11, encodes a serine/threonine kinase. LKB1 plays crucial roles in cell differentiation, proliferation, and polarity. In this study, LKB1 conditional knockout mice (LKB1^Pax2 CKO mice) were generated using Pax2-Cre mice to investigate the function of LKB1 in inner ear hair cells during early embryonic period. LKB1^Pax2 CKO mice died perinatally. Immunofluorescence and scanning electron microscopy revealed that stereociliary bundles in LKB1^Pax2 CKO mice were clustered and misoriented, respectively. Moreover, ectopic distribution of kinocilium bundles resulting from abnormal migration of kinocilium was observed in the mutant mice. The orientation of stereociliary bundles and the migration of kinocilia are critical indicators of planar cell polarity (PCP) of hair cells. LKB1 deficiency in LKB1^Pax2 CKO mice thus disrupted hair cell planar polarity during embryonic development. Our results suggest that LKB1 is required in PCP formation in cochlear hair cells in mice.

Keywords LKB1 stereociliary bundles kinocilium planar cell polarity hearing mice

Corresponding Author(s): Jian Zhang,Jiangang Gao

Just Accepted Date: 08 November 2016 Online First Date: 25 November 2016 Issue Date: 01 December 2016

Cite this article:

Yuqin Men,Aizhen Zhang,Liwen Zhang, et al. Loss of liver kinase B1 causes planar polarity defects in cochlear hair cells in mice[J]. Front. Med., 2016, 10(4): 481-489.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-016-0494-3
https://academic.hep.com.cn/fmd/EN/Y2016/V10/I4/481

Fig.1 Pattern of LKB1 expression in cochlea of mice at E14.5 and P1. (A) Confocal images of cochlea in E14.5 wild-type mice. At E14.5, LKB1 was expressed in nearly all cell types, including hair cells. Scale bar: 50 mm. (B) Confocal images of cochlear hair cells in wild-type mice at P1. LKB1 was expressed in cochlear hair cells of the inner ear. Red: phalloidin, a Factin-specific dye; green: LKB1; and blue: DAPI. Scale bar: 20 mm.

Fig.2 Generation of LKB1^Pax2 CKO mice. (A) Mouse genotyping through PCR analysis. Lanes: wild type (+/+), heterozygous (+/-), and homozygous (-/-) mice. The PCR product of the LKB1 gene in the wild-type mice was 415 bp. In homozygous mice, the PCR product of the LKB1 gene was observed as a single 800 bp band. Both 415 and 800 bp bands were detected in the heterozygous mice. The product of the Pax2-Cre recombinase gene was 500 bp. (B) Western blot analysis of the LKB1 protein in the cochlea of wild-type mice (+ /+) and LKB1^Pax2 CKO (-/-) mice at E17.5. LKB1 protein level was significantly lower in the LKB1^Pax2 CKO mice than in the wild-type mice. n = 3 animals per genotype.

Fig.3 Gross morphology of the wild type and LKB1^Pax2 CKO mice. (A) Overall morphology of wild type and LKB1^Pax2 CKO mice at P0. Scale bar: 1 mm. (B) Overall morphology of wild type and LKB1^Pax2 CKO mice at E17.5. A LKB1^Pax2 CKO mouse showed a pale body and was smaller than the controls. Scale bar: 1 mm. (C) The temporal bones of the LKB1^Pax2 CKO mice are smaller than those of the wild-type mice. (D) Cochlea was shorter in the LKB1^Pax2 CKO mice than in the wild-type mice.

Fig.4 Abnormal cellular patterning of cochlear hair cells in LKB1^Pax2 CKO mice. (A) Confocal images of phalloidin-stained whole mount of the middle turn of cochlear hair cells of wild type and LKB1^Pax2 CKO mice at E17.5. Hair cells in wild-type mice showed regular alignments. The mutant hair cells, especially OHCs, in the LKB1^Pax2 CKO mice showed cellular disorganization and irregular alignments. The degree of disorganization is presented in the inset on the left. Green: phalloidin, an F-actin-specific dye. Scale bar: 20 mm. (B) Phalloidin-stained whole mount of the middle turn of cochlear hair cells of wild type and LKB1^Pax2 CKO mice at E17.5. Regular alignment of hair cells in wild-type mice was observed from the images of the stained whole mounts. Disorganized cell alignment was found in LKB1^Pax2 CKO mice. The degree of disorganization is shown in the inset on the left. Green: phalloidin. Scale bar: 20 mm.

Fig.5 Dysmorphology and misorientation of stereociliary bundles of cochlear hair cells in LKB1^Pax2 CKO mice. (A) Confocal images of phalloidin-labeled stereocilia of the wild type and LKB1^Pax2 CKO mice at P0. The cochlear hair cells in the wild-type mice exhibited an organized, stair-like, V-shaped morphology and displayed uniform orientation of stereociliary bundles. The stereociliary bundles of hair cells in mutant mice showed clustered morphology and disrupted orientation. Red: phalloidin. Scale bar: 5 mm. (B) SEM images showing clustered (white arrow) and misoriented stereociliary bundles in hair cells of mutant mice compared with the uniform organizations in wild-type mice. High-magnification SEM images show the orientation of stereociliary bundles in the wild type and LKB1^Pax2 CKO mice (yellow arrows). The vertices of stereociliary bundles in the wild-type mice point in one direction, whereas the vertices in the mutant mice point in different directions. Scoring of the orientation of hair cell bundles is shown on the left panel of the diagrams. HCs: hair cells; OHCs: outer hair cells; IHCs: inner hair cells. Scale bar: 5 mm. (C) Statistical analysis of the angular orientations of stereociliary bundle in wild type and LKB1^Pax2 CKO mice at E17.5. Distribution of the variously rotated stereociliary bundles in hair cells is shown. The hair cell bundles in wild-type mice showed uniform orientations (upper panel). By contrast, the cochlear hair cell bundles in LKB1^Pax2 CKO mice showed irregular orientations (lower panel). The distribution of the stereociliary bundle with angular orientation was wider in the mutant mice than in the wild-type mice. n = 4 animals per genotype. Data are presented as percentages.

Fig.6 Abnormal positioning of kinocilia in cochlear hair cells of LKB1^Pax2 CKO mice. (A, B) Cochlear hair cells were stained with anti-acetylated tubulin antibody, a marker for kinocilium. (A) In wild-type mice, kinocilia of hair cells were arranged uniformly at the apical surface of the hair cells (white arrows). (B) In LKB1^Pax2 CKO mice, the mispositioned kinocilia were found at the vertex of the V-shaped misoriented stereociliary bundles (white arrowheads). Red: phalloidin; green: acetylated tubulin. Scale bars: 20 and 10 mm. (C, D) Cochlear hair cells were stained with anti-g-tubulin antibody to label the basal region of kinocilium. (C) In wild-type mice, the basal bodies of kinocilia were aligned uniformly at the apical surface of the hair cells (white arrows). (D) In the LKB1^Pax2 CKO mice, the basal bodies of kinocilia were disordered (white arrowheads). Red: phalloidin; green: g-acetylated tubulin. Scale bars: 20 and 5 mm.

Fig.7 Mispositioning of kinocilia of cochlear hair cells in LKB1^Pax2 CKO mice. Scanning electron micrograph of hair cell bundles in the wild type and LKB1^Pax2 CKO cochlea at E17.5. In wild-type mice, kinocilium (green dots) is uniformly located at the lateral periphery of the apical surface of hair cells. The kinocilium was found at the vertex (red dots) of the V-shaped stereociliary bundle. However, in the mutant mice, kinocilium (green dots) was mispositioned at the lateral cell edge of hair cells. In most hair cells, the kinocilia (green dots) remained at the vertex (red dots) of the misoriented stereociliary bundles. Only in few outer hair cells, the kinocilia (green dots, arrows) were deviated from the vertex (red dots) of the V-shaped stereociliary bundles. The positions of the kinocilia and the vertex of the V-shaped stereociliary bundles were marked with green and red dots, respectively. HCs: hair cells; OHCs: outer hair cells; and IHCs: inner hair cells. Scale bar: 5 mm.

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