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

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

Introduction

The innermost part of the vertebrate ear is the inner ear, which is mainly responsible for sound detection and balance. Cochlea is one of the auditory parts of the mammalian inner ear. Its core component is the organ of Corti (OC), which is the sensory organ for hearing. The OC in mice consists of a single row of inner hair cells (IHCs) and three rows of outer hair cells (OHCs) [ 1]. Structures on the apical surface of each hair cell include hair bundles, which can be divided into two types: actin-based stereociliary bundles and single tubulin-based kinocilium [ 2, 3]. Each stereociliary bundle, which is a mechanosensor, is organized into a V-shaped, ladder-like structure. In mice, staircase formation and functional maturation of a hair bundle occur in the first two weeks after birth, and the hair bundle is maintained at a constant length throughout the lifespan of a mouse. The longest stereocilia is juxtaposed next to an axonemal cilium, the kinocilium. The kinocilium is located in the vertices of hair bundles, points away from the center of the cochlea, and retracts approximately two weeks after birth. Hair bundle development and maturation is a complex and precisely regulated process. These intrinsic structural features of hair bundles are essential for the correct perception of sound during hearing process [ 4– 6].

Liver kinase B1 (LKB1) gene is an important serine/threonine kinase 11 (STK11) and a potent tumor suppressor [ 7]. LKB1 was identified and characterized in a region of chromosome 19p13.3, which is a locus for Peutz–Jeghers syndrome. LKB1 encodes a 48 kDa protein, which is ubiquitously expressed in fetal tissue, particularly in pancreas, liver, testes, and skeletal muscles. High LKB1 gene expression in conventional LKB1 gene knockout mice results in embryonic lethality at approximately 8‒9 embryonic days (E), and the mice display a variety of developmental abnormalities [ 8, 9]. LKB1 conditional knockout models were established to thoroughly study the function of LKB1 protein during embryonic development and during adult life. Experiments using these knockout mouse models found that LKB1 plays crucial roles in cell polarity, cell cycle regulation, and cell growth and differentiation in developing mice [ 9– 13].

However, although a number of studies on LKB1 has been performed, few of them have elucidated the functions of LKB1 in regulating the development of inner ear hair cells in mice. In our previous study on inner ear, an LKB1 conditional knockout mouse model (LKB1^Atoh1 CKO mice) was created by crossing LKB1^LoxP/LoxP and Atoh1-Cre mice, which expressed Cre in the inner ear at E13.5 [ 14]. The LKB1^Atoh1 CKO mice displayed impaired hearing and malformed stereociliary bundles of cochlear hair cells after birth. We speculate that LKB1 plays an essential role in the development and maintenance of stereocilia in inner ear hair cells in mice [ 14]. These studies have shown that LKB1 plays a role in controlling hair cell formation at late developmental stages, that is, after E13.5; however, some clarifications, especially on the role of LKB1 in some critical early stages of hair cell development, must be made.

In mice, the development of inner ear and hair cells proceeds through several critical stages. The inner ear begins to develop from oticplacode at E8. At E9, the oticplacode forms into an otocyst. In mice, all otocyst cells, including auditory progenitors, continue to divide from E9 to E12 [ 15– 17]. Outgrowth of the cochlea and the vestibule from the otocyst appear at around E11 in mice. Auditory progenitors withdraw from the cell cycle at E12 [ 18]. At E13, the auditory progenitors start to differentiate into HCs and supporting cells [ 15, 18– 20]. With the initiation of hair cells, hair bundles develop at this stage. All of these significant events in hair cell development, including formation of oticplacode, proliferation of auditory progenitors, initiation of hair cells, and differentiation of auditory progenitors, occur between E8 and E13. Atoh1-Cre expresses Cre recombinase at E13.5 [ 21]. Cre protein usually requires some time to accumulate and to become functional to excise its target DNA. Thus, the role of LKB1 in early embryonic development, especially in some critical stages that occur between E8.5 and E13.5 as mentioned above, cannot be studied in LKB1^Atoh1 CKO mice.

To investigate the function of LKB1 in early embryonic development of hair cells, we created LKB1^Pax2 CKO mice by crossing LKB1^LoxP/LoxP mice with Pax2-Cre mice that express Cre in their inner ear at E7‒8 [ 22]. The LKB1^Pax2 CKO mice showed disoriented stereociliary bundles and an ectopic kinocilium, indicating that loss of LKB1 in early hair cell development leads to defects in planar polarity of cochlear hair cells [ 22].

Materials and methods

Animals

To establish LKB1^Pax2 CKO mice, we crossed mice homozygous for “floxed” LKB1 allele (LKB1^LoxP/LoxP) (MGI accession number: 4440829) [ 23] with Pax2-Cre mice [ 22]. Pax2 gene is expressed in developing otocyst, kidney, midbrain–hindbrain boundary, and inner ear [ 22]. Moreover, Pax2-Cre activities are detected in CO, Reissner’s membrane, renal pelvis, and stria vascularis in inner ear [ 22]. For timed pregnancies, the morning of the positive plug check was considered E0.5, and the day of birth was designated postnatal day 0 (P0).

Animal management and use were strictly in accordance with the standards of the Animal Ethics Committee of Shandong University. All procedures of our animal experiment were approved by the Ethics Committee of Shandong University.

PCR genotyping

DNA was extracted from tail snips of mice for PCR analysis. The mice were examined for Cre through PCR by using the following primers: Cre-F (5′-AGCTAAACATGCTTCATCGTCGGTC-3′) and Cre-R (5′-TATCCAGGTTACGGATATAGTTCATG-3′). A 500 bp band indicated the presence of Cre allele.

PCR genotyping of LKB1^LoxP/LoxP was performed using the following primers: LKB1-F (5′-ATCGGAATGTGATCCAGCTT-3′) and LKB1-R (5′-GAGATGGGTACCAGGAGTTGGGGCT-3′). A 415 bp band was detected in the wild-type mice. Bands of 415 and 800 bp were detected in heterozygous mice. Only the 800 bp band was observed in the homozygous mice.

Immunofluorescence analysis

Immunofluorescence staining was performed as described previously [ 14]. Briefly, inner ears obtained from the mice were fixed overnight in 4% paraformaldehyde at 4 °C. For sectioning, the cochlea samples were equilibrated in 15% sucrose and then in 30% sucrose overnight at 4 °C. The samples were embedded in OCT compound and then sectioned into 7 mm thickness. For whole mount immunostaining, the cochleae were dissected to expose the sensory epithelium. The sensory epithelium was divided into basal, middle, and apical sections. The samples were washed with 10 mmol/L PBS and stained with rhodamine phalloidin (2 µg/ml, Sigma) for 30 min at room temperature (RT) or with primary antibody overnight at 4 °C followed by three washes in PBS for 10 min each round. After three washes, the samples stained with primary antibody were incubated in secondary antibodies diluted in a solution of 0.1% bovine serum albumin and 0.1% Triton X-100 at 37 °C for 1 h. The samples stained with antibodies were washed with 10 mmol/L PBS and then incubated in rhodamine phalloidin (2 µg/ml, Sigma) and nuclear stain for 15 min at RT followed by final washing in 10 mmol/L PBS. The samples were analyzed under a laser scanning confocal microscope (LSM780). The primary antibodies used were anti-LKB1 polyclonal antibody (rabbit, 1:200, Upstate), anti-acetylated tubulin monoclonal antibody (mouse, 1:500, Sigma), and anti-g-tubulin monoclonal antibody (mouse, 1:500, Sigma).

Western blot analysis

Western blot analyses were performed as described previously [ 14]. Cochleae of inner ears were obtained from the mice at E17.5. Cochlear protein was incubated in cell lysis buffer (10 mmol/L Tris, pH= 7.4, 1% TritonX-100, 150 mmol/L NaCl, 1 mmol/L EDTA, and 0.2 mmol/L PMSF) and then extracted using a glass grinding rod. The cell lysis buffer containing cochlear protein was subsequently centrifuged at 1200 r/min for 20 min at RT. The protein from the samples (40 mg) was subjected to SDS-polyacrylamide gel electrophoresis and then blotted onto a polyvinylidene difluoride membrane.

The primary antibodies used in our study are anti-LKB1 polyclonal antibody (Rabbit, 1:500, Bioworld) and anti-GAPDH monoclonal antibody (mouse, 1:5000, Millipore).

Scanning electron microscopy (SEM)

For SEM analysis, inner ears obtained from mice at E17.5 were dissected in PBS and fixed overnight with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4) at 4 °C. The cochleae were subsequently dissected to expose the sensory epithelium. The sensory epithelia were washed three times in PBS, post-fixed for 1 h with 1% osmium tetroxide, and then dehydrated through a graded ethanol series ranging from 30% to 100%. The specimens were critical-point dried, mounted on metal studs, and sputter coated with gold. After all the treatments, the morphology of each hair bundle was examined, and images were taken using a Hitachi S-4800 Field-Emission Scanning Electron Microscope. SEM was performed as described previously [ 14].

Results

Conditional inactivation of LKB1 by Pax2-Cre in developing inner ear

To determine the distribution of LKB1 protein in the inner ear, we immunostained the cryosections with an anti-LKB1 antibody at E14.5 (Fig. 1A). As early as E14.5, LKB1 proteins were present in nearly all of the cochlea cells in mice (Fig. 1A). Whole mount immunostaining was used to examine LKB1 expression in the cochlear hair cells at P1. The results showed high LKB1 expression in hair cells of the wild-type mice (Fig. 1B).

To generate LKB1 conditional knockout mice, we crossed LKB1^LoxP/LoxP mice with Pax2-Cre mice to specifically delete LKB1 gene from the inner ear hair cells. The LKB1 exons 3–6 flanked by LoxP sites were removed upon Cre expression in LKB1^Pax2 CKO mice. The wild-type littermates of the knockout mice were used as controls. The genotypes of the pups were identified by PCR analysis (Fig. 2A). LKB1 protein level in the mice was examined at E17.5 through Western blot analysis. The results showed that LKB1 protein levels were significantly lower in the entire cochlea of the homozygous knockout mice (Fig. 2B) than in the cochlea of the controls. These results revealed that LKB1 was conditionally knocked out in the inner ear of LKB1^Pax2 CKO mice.

The LKB1^Pax2 CKO mice died perinatally. We sacrificed the pregnant female mice and dissected the LKB1^Pax2 CKO embryos at E17.5. The death rate of LKB1^Pax2 CKO mice at this stage was approximately 30% (data not shown). LKB1 ^Pax2 CKO mice displayed pale bodies because of lack of blood (Fig. 3A and 3B). We dissected the cochlea of the LKB1^Pax2 CKO mice and found that they were smaller than those of the littermate controls (Fig. 3C and 3D).

LKB1 deletion in LKB1^Pax2 CKO mice resulted in alignment defects in hair cells and malformation of hair cell stereocilia

To investigate the function of LKB1 in cochlear hair cells during embryonic development, we stained cochlear whole mount with rhodamine phalloidin and then performed SEM analysis to examine the morphology of hair cells and their stereociliary bundles in both LKB1^Pax2 CKO mice and wild-type mice at E17.5. Uniformly aligned hair cell rows were observed in the controls (Fig. 4). By contrast, the LKB1^Pax2 CKO mice showed disordered alignment of hair cells (Fig. 4). These results demonstrated that LKB1 deficiency caused hair cell patterning defects in LKB1-deficient mice (Fig. 4). Additionally, the number of hair cells in the LKB1^Pax2 CKO mice did not significantly differ from that in the wild-type mice.

In wild-type mice, regular V-shaped stereociliary bundles with vertices pointing in one direction were uniformly oriented across the epithelium (Fig. 5A and 5B). In LKB1^Pax2 CKO mice, abnormal bundles were found in the OHCs. The stereociliary bundles in the mutant OHCs showed an irregular and clustered patterning (Fig. 5A and 5B). Moreover, the vertices of stereociliary bundles pointed in different directions, indicating misorientation of the bundles (Fig. 5A and 5B). The orientation of mutant IHC bundles in LKB1^Pax2 CKO mice showed minor abnormalities (Fig. 5A and 5B). Fig.5C shows the quantification of the orientation of stereociliary bundle at E17.5 in the control and LKB1^Pax2 CKO mice. These results demonstrated misorientation of stereociliary bundles of hair cells in LKB1^Pax2 CKO mice.

LKB1 inactivation led to mispositioning of kinocilium in hair cells in LKB1^Pax2 CKO mice

The structure and position of kinocilia were examined through immunostaining, wherein the kinocilia were stained with the kinocilium markers acetylated tubulin and g-tubulin. In the wild-type mice, the kinocilia were uniformly aligned at the lateral periphery of the apical surface of hair cells (Fig. 6A and 6C, arrows). The kinocilium was located at a fixed position at the apical surface of hair cells (Fig. 6A and 6C, arrows). By contrast, the kinocilia in the LKB1^Pax2 CKO mice were disorganized and mispositioned at the lateral periphery of the apical surface of hair cells (Fig. 6B and 6D, arrowheads).

To visualize kinocilia in detail, we examined the kinocilia in cochlear hair cells of wild type and mutant mice through SEM. In wild-type mice, the kinocilia were located at the vertex of the V-shaped stereociliary bundles and at a fixed position on the lateral periphery of the hair cell apical surface (Fig. 7). In most hair cells of the LKB1^Pax2 CKO mice, kinocilia remained at the vertex of the V-shaped bundles. However, because of the misorientation of the stereociliary bundles, these kinocilia were mispositioned at the lateral periphery of the hair cell apical surface (Fig. 7). In few OHCs of the mutant mice, the kinocilia were deviated from the vertex of V-shaped stereociliary bundles (Fig. 7).

Discussion

Being an important serine/threonine kinase, LKB1 plays a significant role in regulation of cell differentiation, migration, growth, and apoptosis and in the establishment of cell polarity during development in mice [ 9– 13, 24]. LKB1 plays a crucial role in multiple development signaling pathways and in the regulation of cellular life cycle. Although studies have demonstrated the various crucial functions of LKB1, the functions of LKB1 in hair cells and stereociliary bundles remain poorly understood.

In our study, we created a new LKB1 conditional knockout mouse model by crossing LKB1^LoxP/LoxP mice with Pax2-Cre mice, which express Cre recombinase in their inner ear hair cells beginning at E7‒8 [ 8, 9]. We investigated the functions of LKB1 in some critical stages of early embryonic development of inner ear hair cells in mice. We observed that the proper alignment of cochlear hair cells was disrupted in LKB1^Pax2 CKO mice. Moreover, immunostaining and SEM showed the clustered and misoriented stereociliary bundles, as well as the mispositioned kinocilium atapical surface of inner ear hair cells in LKB1^Pax2 CKO mice. These results suggested that LKB1 deficiency caused evident abnormalities during development of inner ear hair cells in mice and that these abnormalities particularly affected the hair cell bundles.

In mice, stereociliary bundles at the apical surface of hair cells comprise rows of actin-based, V-shaped stereociliary bundles and a single specialized primary kinocilium [ 2, 3]. The kinocilium is located at the vertex of the V-shaped stereociliary bundle [ 25, 26]. The vertex of the V-shaped stereociliary bundles located on the apical surface of hair cells uniformly points toward the lateral edge of the cochlear duct. Development and maturation of hair bundles in the inner ear is a complex and precisely regulated process. During embryogenesis of the inner ear, the kinocilium starts to develop at the center of the hair cell apical surface, migrates in a specific direction from the center to the lateral periphery of the cell, and then becomes localized at a fixed position in the apical surface [ 27]. The kinocilium in LKB1^Pax2 CKO mice was mispositioned at apical surface of the hair cell. Therefore, we speculate that LKB1 is required for the correct positioning of kinocilia in mice. Being a specialized tubulin-based cilium, the kinocilium is speculated to guide the morphogenesis of hair cell bundles [ 27]. The morphology of hair cell bundles depends on the proper development and migration of kinocilium [ 25, 26, 28, 29], which determines the orientation of stereociliary bundles [ 25, 26]. Moreover, we observed clustered and misoriented stereociliary bundles in inner ear hair cells of LKB1^Pax2 CKO mice. This result is consistent with previous reports, in which dysmorphic hair cell bundles are often associated with abnormal positioning of kinocilium in cochlear hair cells.

The positioning of kinocilium and the uniform orientation of hair bundles in the inner ear are controlled by the planar cell polarity (PCP) pathway [ 30– 33]. PCP is crucial in epithelial morphogenesis and plays a role in the development of inner ear hair cells in mice [ 34]. The morphological features of stereociliary bundles and kinocilium contribute to the planar polarity of hair cells [ 28– 30, 33]. In mice, planar polarity of hair cell is established during embryogenesis. PCP of hair cells is initially generated during hair cell formation [ 30]. During hair cell development, the earliest event in hair cell planar polarization is the migration of the kinocilium from the center of the apical surface toward the lateral periphery of the hair cells. Concomitant with the migration of kinocilium, microvilli in the apical surface of hair cells begin to form stereocilia with a regulated V shape and graded heights. The V-shaped stereociliary bundles showed a uniform orientation on the apical surface of the hair cells, and this feature is a significant marker of PCP. In PCP mutants, hair bundles are frequently misoriented relative to the mediolateral axis of cochlea [ 35]. In our study, mispositioned kinocilia and misoriented stereociliary bundles were observed in LKB1^Pax2 CKO mice, indicating that LKB1 deficiency disrupted the PCP during hair cell development. Our findings suggest that LKB1 is required for the establishment of proper PCP of hair cells in mice.

The mechanism by which LKB1 regulates PCP establishment in cochlear hair cells remains unclear. Studies have shown that LKB1 directly phosphorylates the serine/threonine kinase PAR1A, thereby regulating its kinase activity. PAR1A then activates Dishevelled (Dvl), a component of the Wnt pathway [ 8, 36]. Thus, LKB1 indirectly regulates the Dvl by phosphorylating PAR1A. In addition to regulating Dvl, LKB1 regulates the transcription of several genes, including Frizzled2, which are involved in Wnt signaling [ 37]. These Wnt signaling pathway core proteins, namely, Dvl and Frizzled proteins, can regulate PCP during development in mice [ 38– 42]. Therefore, we speculate that LKB1 controls the PCP of hair cells through Wnt signaling pathway in mice. Our studies propose a new perspective on PCP control, that is, PCP is controlled by LKB1 in the early embryonic development of hair cells in mice.

Being an important serine/threonine kinase in vivo, LKB1 is involved in various human diseases, including Peutz‒Jeghers syndrome. Given the severe malformation of hair cell bundles in the inner ear of LKB1-deficient mice, Peutz−Jeghers patients may suffer from impaired hearing, and hearing screening in such cases is warranted. Additionally, hearing-impaired people can be screened for LKB1 mutations. Not only can the LKB1^Pax2 CKO mice be used to examine LKB1 gene functions during development and maturation of the hearing system but they can also provide an animal model that can be used to explore therapeutic interventions for deafness in humans.

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