The Function Of Lkb1 Gene In The Development Of Inner Ear And Cerebellum In Mice

BackgroundThe LKB1 gene, also known as STK11, is a serine/threonine protein kinase. It is a tumour suppressor, and it has an important association with the Peutz-Jeghers syndrome (PJS). PJS is an autosomal dominant genetic disease with clinical manifestations including multiple hamartoma-like gastrointestinal polyps, mucocutaneous pigmentation, and a high degree of tumour susceptibility. In recent years, LKB1 gene was recognized as the major causative gene of PJS. However, due to the complexities of the pathogenesis caused by mutations in the LKB1 gene, its biological role has not been fully elucidated. In 2002, to further determine whether LKB1 is a key pathogenic gene in PJS syndrome and to determine the important biological functions of the LKB1 gene, Jishage et al. created an LKB1 knockout mouse model to solve the problem mentioned above. In homozygous knockout mice, they found that homozygous knockout mice died in mid-gestation at about embryonic days 8.5 to 9.5 (E8.5-9.5), showing embryonic neural tube defects, mesenchymal cell death, and vascular abnormalities. At E9, compared to the littermates, the homozygous knockout mice usually showed a smaller body and retardation in growth. In addition, heterozygous knockout mutant mice revealed multiple gastrointestinal polyps in 10-14 months. These results implied that the Lkbl gene is essential during embryogenesis and development in mice. The Lkbl gene is also closely related with tumorigenesis.Currently, as a tumour suppressor gene, LKB1 has become a gene of great interest. It is expressed in almost all tissues, especially epithelium, stomach, intestine, testis seminiferous tubule and other tissues. Due to the wide range of LKB1 gene expression, conventional knockout of LKB1 results in embryonic lethality, thus greatly hindering research on the biological functions of LKB1 in specific tissues and cells in vivo. In recent years, to further study the role of the LKB1 gene in specific tissues, a large number of conditional knockout mice have been generated and have been studied to determine the important functions of LKB1 in different tissues in vivo. Previous studies have been reported that the LKB1 gene plays an important role in the regulation of cell growth through many signalling pathways, including TGF-β, NF-κB, PTEN/PI3K, G protein-coupled receptors, mTOR and several other signalling pathways. LKB1 is involved in the regulation of many cellular activities, including cell proliferation, apoptosis, the cell cycle, cell polarity, chromatin remodelling and energy metabolism. However, we found that there are still some gaps of Lkbl gene research in the nervous system of mice, such as in the inner ear and cerebellum. Thus, in this paper, we focus on the function of the Lkbl gene in the development of the inner ear and cerebellum in mice.Scientific questions to be solvedIn this thesis, using conditional knockout technology, we obtained two types of LKB1 conditional knockout mice, Pax2-LKB1 knockout mice and Atohl-LKB1 knockout mice by conditional knock-out technology. Using two conditional knock-out models, we studied the function of the Lkbl gene in the inner ear and in the cerebellum in mice. Scientific issues to be solved are as follows:(1) The function of LKB1 in the development of inner ear hair cells in mice; (2) The molecular mechanism of LKB1 in the development of the inner ear hair cells; (3) The function of LKB1 in the development and foliation of the cerebellum in mice; (4) The molecular mechanism of LKB1 in the development and foliation of the cerebellum.Research programs and resultsIn the thesis, to solve the scientific questions mentioned above, we created the LKB1 conditional knockout mice, Pax2-LKB1 conditional knockout mice. In the thesis, we firstly studied the function and molecular mechanism of LKB1 in the development of cochlear hair cells in the inner ear during the early embryonic stage in mice. The Pax2-LKB1 conditional knockout mice expressed Cre recombinase in the inner ear at E7-8. In this study, we observed that some of Pax2-LKB1 conditional knockout embryos died perinatally, and the others survived at E18.5. At a gross level, Pax2-LKB1 conditional knockout mice showed pale body lacking blood. To investigate the function of LKB1 in cochlear hair cells during embryomic development, cochlear whole mounts staining with rhodamine phalloidin, a specific marker for stereociliary bundles was used to examine the morphology of stereociliary bundles. In wild type mice, regular V-shaped stereociliary bundles were uniformly oriented across the epithelium. In the Pax2-LKB1 conditional knockout mice, abnormal bundles were found in the outer hair cells. The stereociliary bundles in the mutant OHCs showed a clustered patterning and misorientation of the bundles. Next, to investigate structure of kinocilia defects, we double stained with acetylated tubulin, as a kinocilium marker, and phalloidin was used to display the position of the kinocilium relative to the stereociliary bundles. In the wild type mice, kinocilium was uniformly positioned at the lateral periphery of hair cells apical surface. The kinocilium was located at fixed position at hair cells apical surface. However, In the Pax2-LKB1 conditional knockout mice, the positions of these kinocilium was disorganized and dislocated at the lateral periphery of hair cells apical surface in hair cells. To examine the structure of hair cell bundles in detail, we examined the hair cell bundles in cochlear hair cells of wild type and mutant mice by scanning electron microscope. The result of SEM also showed clustered and misoriented the stereociliary bundles in inner ear hair cells of the Pax2-LKB1 conditional knockout mice. Additionally, in the Pax2-LKB1 conditional knockout mice showed kinocilia were still at the vertex of the V-shape in the most of hair cells, but these kinocilia were dislocated at the lateral periphery of hair cells apical surface. We observed that the kinocilia were deviated at the vertex of V-shaped stereociliary bundles in only a few outer hair cells of the mutant mice. During the development of the hair cell bundles, the kinocilium is crucial for determing the orientation of stereociliary bundles. The proper orientation of stereociliary bundles is a critical symbol for planar cell polarity (PCP) of hair cells. Thus, we propose that LKB1 is required for the establishment of proper planar cell polarity of hair cells in mice.Because Pax2-LKB1 conditional knockout mice displayed perinatal death, we could not further study the function of the Lkbl gene in the inner ear. Thus, we created another conditional knockout mouse, Atohl-Cre mice to specifically delete the Lkbl gene in the inner ear at E13.5-14.5. Because the expression time of Atohl-Cre was later than that expressed in Pax2-Cre, we obtained the Atohl-LKB1 conditional knockout mice that are viable and fertile. Thus, we further studied the function and molecular mechanism of the Lkbl gene in the development and maturation of inner ear hair cells through the analysis of the Atohl-LKB1 conditional knockout mice.In our study, there were no significant difference between the mutant mice and the controls in gross morphology, except for the decreased weight in the mutants. However, the hearing thresholds including the ABR and DPOAEs in LKB1 knockout mice were significantly higher than those in the control mice. By immunostaining with phalloidin, the analysis of the hair cell stereociliary bundles in mice’s inner ears at P1 showed minor abnormalities in both OHCs and IHCs. However, at about P7, the outer hair cell stereociliary bundles displayed a conformational "U" shaped abnormality and irregular stair-like structure. After P7, the morphogenesis of stereocilia was more severely damaged in the outer hair cell stereociliary bundles of the mutant group. In our results, we also observed that the sporadic loss of hair cells in LKB1-deficient mice starting at P14. Due to the morphological appearance in chronological order of the disordered stereociliary bundles before hair cell loss, we speculate that there is a direct causal link between the LKB1 gene deficiency and stereociliary bundle abnormalities, resulting in the sporadic loss of hair cells finally. In mice, stereociliary bundles in inner ear hair cells are staircase cytoskeletal microvilli-structures with actin filaments at the core and several actin-binding proteins, such as ERM (ezrin/radixin/moesin), villin, fimbrin, XIRP2, and FASCIN2. These actin-binding proteins participate in the assembly of highly organized actin-based structures and maintain the stability of the stereocilia during the development of hair cells. Among these actin-binding proteins, we first focused on the significant ERM protein family, which is related to bundling actin filaments in the development of hair cell stereocilia. We first examined ERM protein expression and determined that ERM is primarily enriched in the stereocillia of hair cells in mice. Next, the expression level of ERM and pERM were tested in mutant and wild-type mice by Western blot. We found a small increase in the total expression level of ERM in the mutant mice. We proposed that, in our mutant mice, the significant decrease of pERM levels potentially resulted in the failure to regulate the actin assembly. Previous studies have demonstrated that LKB1 can act as the upstream regulator protein to indirectly phosphorylate the actin-binding protein Ezrin, a member of ERM proteins through Mst4, and the pERM induced brush border formation in the intestinal epithelial cell. The structure of stereocilia is similar to that of the brush border, and both of their primary components are the actin-based cytoskeleton. Thus, we investigated whether the Mst4 was affected by the LKB1 deletion in the Atoh1-LKB1 conditinal knockout mice. We first examined the Mst4 protein expression in cochlea hair cells. By immunostaining analysis, we found that Mst4 is highly expressed in hair cells. By Western blot analysis, we found that the deletion of LKB1 caused a decreased amount of Mst4 in the Atoh1-LKB1 conditinal knockout cochlea. It is likely that Mst4 acts an intermediate protein between LKB1 and ERM to phosphorylate ERM during the development and maintenance of stereocilia in hair cells. Taken together, during the development of hair cells, LKB1 may regulate the development and maintenance of stereocilia via the activation of ERM to ensure the function of hearing.The Atohl-Cre activities were confined to not only the developing inner ear but also the cerebellum. Thus, we chose the Atohl-LKB1 conditional knockout mice to further study the function of the Lkbl gene in cerebella of mice. In this thesis, we focus on the function and molecular mechanism of LKB1 in the development and foliation of the cerebellum in mice.The cerebellum is a critical motor organ that controls both motor coordination and motor learning and also plays a critical role in cognition, affect and behaviour. In our thesis, behavioral tests including gait test, rotarod test, and tightrope test indicated that the mutant mice suffered motor dysfunction. By immunohistochemistry and immunostaining, we found that the overall structure had a larger volume and morelobules in the Atoh1-LKB1 conditional knockout cerebellum. It is reported that there is closely relation between cerebellar folation and the proliferation of GCPs. We examined the proliferation of GCPs labelled with anti-phospho-histone H3 (PH3) antibodies, a marker of mitotic cells, or anti-BrdU to measure GCP proliferation. Compared to the wild type mice, LKB1 inactivation in the Atoh1-LKB1 conditional knockout mice led to an increased proliferation of granule cell precursors (GCPs). We examined the migration of GCs by BrdU pulse chase experiments to investigate if the GCP migration was disrupted by LKB1 deficiency.96 h after the BrdU pulse, most of BrdU+cells in the control cerebellum already passed the ML and PCL and reached the IGL. In contrast, much less BrdU+cells were observed in the IGL in the Atohl-LKB1 conditional knockout mice compared with the wild-type mice. Thus, the loss of LKB1 not only caused increased proliferation of GCPs but also affected GCP migration during the cerebellar development in mice. Additionally, in this study, we also found aberrant Purkinje cells development in the Atohl-LKB1 conditional knockout mice. These phenotypes indicate that LKB1 is essential for mouse cerebellar development. It was reported that sonic hedgehog (Shh) is responsible for the rapid GCP proliferation. Shh signalling is mediated by the Gli family of transcription factors. In the Atohl-LKBl conditional knockout cerebellum, the expression levels of the Gli genes were significant increased, indicating elevated Shh signalling. Cyclin D1 is a direct target of the Shh pathway and functions to regulate cell cycle progression in granule cell precursors by increasing G1 cyclin expression. The expression of cyclinDl was significantly increased in the early postnatal Atohl-LKBl conditional knockout cerebellum. Thus, these results revealed that LKB1 deletion may promote GCP proliferation via elevated Shh signalling and cyclinDl expression in the developing cerebellum. In vitro proliferation assays were performed to verify whether LKB1 regulates GCP proliferation through Shh signalling. In consistency with the data from in vivo experiment results, our in vitro results support the notion that LKB1 may regulate the proliferation of GCPs via sonic hedgehog signalling. Previous reports demonstrated that AMPK can be phosphorylated by LKB1 and can also negatively regulate Shh signalling by interacting with Gli1 in vitro. In our study, we examined the expression of AMPK by Western blot and found that the mutant cerebellum had similar total AMPK levels and the significantly decreased p-AMPK. Thus, AMPK likely acts as an intermediate protein between LKB1 and Shh signalling in the developing cerebellum. Thus, LKB1 regulates cerebellar development and foliation by controlling Sonic Hedgehog-mediated granule cell precursor proliferation and granule cell migration.SignificanceIn this thesis, we studied the important function of the Lkbl gene in the inner ear and cerebellum during mouse development. At the molecular and cellular level, we also reveal the developmental mechanisms of LKB1 in the inner ear and cerebellum to further provide a theoretical basis in nervous system development. In addition, diseases in nervous system are among the most common diseases and are known to be difficult to cure in the medical community. In the human nervous system, there are many diseases in the inner ear and the cerebellum, including deafness, movement disorders, hypogenesis of the inner ear and cerebellar vermis. However, the diagnosis and pathogenesis of these diseases still need to be studied.In this thesis, Atohl-LKB1 conditional knockout mice are viable and fertile and can act as a mouse model of human diseases to provide a theoretical basis for clinical diagnosis and treatment. The Atohl-LKB1 conditional knockout mouse model is also important for clinical treatment. Our results indicate that LKB1 plays an important role in the development and maintenance of stereocilia in cochlea hair cells. These findings may provide new research directions for the treatment of hearing loss and some diseases of the cerebellum. In addition, in patients who are diagnosed with Peutz-Jegher Syndrome, screening for LKB1 mutation could reveal a genetic disposition for the condition, and also provide an ideal mouse model to study therapeutic intervention for human deseases. Therefore, our results not only provide clues to the molecular mechanisms of the inner ear and the cerebellum, but also promote the development of the inner ear and the cerebellum as important to the clinical diagnosis and treatment of disease.