Histone Deacetylase Activity Is Necessary For The Early Development Of The Posterior Lateral Line System In Zebrafish (Danio Rerio) Larvae And The Differentiation Of Cochlea Progenitor Cells

The sensory hair cells of the inner ear are highly metabolic and are sensitive to damage. Consequently, the major cause of sensorineural hearing loss in human are attributed to hair cells loss. Because the mammalian inner ear fails to regenerate or repair lost hair cells, leading to permanent and irreversible hearing loss. In contrast, auditory hair cells of nonmammalian vertebrates can regenerate naturally. As a very powerful model system, zebrafish (Danio rerio) offers several distinct advantages over mammalian models. The larva is optically transparent, making easily developed fluorescent transgenic zebrafish and visualized for live imaging during early embryogenesis. The hair cell within lateral-line-neuromast closely related to those in mammalian inner ear in both structure and function. Lateral line system are superficial sensory organs, which make live imaging easily visualized during early embryogenesis and useful to study for drug discovery in vivo. Thus, the lateral line of zebrafish represent a powerful model for identifing molecules mechanism underlying mechanosensory hair cell differentiation and regeneration.Fish and amphibians have a mechanosensory organ called lateral line (LL) related to the auditory system, used to detect directional water flow. The lateral line comprises a set of superficial sense organs called neuromasts, located on the surface of the head (anterior lateral line, ALL) and body (posterior lateral line, PLL) in species-specific patterns. The mature lateral-line-neuromast is composed of a group of hair cells (HCs) in the center and supporting cells (SCs) surrounded at the periphery. Recent studies have been conducted to understand the migration and morphogenesis of PLL system and lateral line hair cells biology relating to mammalian hearing.The acetylation and deacetylation of core histones are the most important histone modifications and determine many cellular processes, including proliferation, apoptosis and differentiation. Histone acetylation is usually associated with transcriptional activation. Conversely, histone deacetylation is associated with transcriptional repression. Histone acetylation and deacetylation is governed by the opposing effects of two enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs), which acetylate and deacetylate specific lysines in the tail residues of histones, respectively. Epigenetics plays an instructive role in a number of biological process, and is yet an essential regulator in the neuronal development and specification processes. However, little epigenetic work has been conducted in explaining development, survival, function and pathology of hearing research.Recent studies suggested that HDACs play positive roles in regulating of supporting cell proliferation in mouse postnatal utricular epithelia. HDAC inhibitors treatment leaded to decreased proliferation of supporting cell from the avian utricle. However, little epigenetic work has been conducted in explaining early development of posterior lateral line (PLL) organ and differentiation of hair cells within lateral-line-neuromast. In this study, we focuses on the possible roles of HDACs in PLL primordium migration, proneuromast deposition and the hair cell differentiation process by using HDAC inhibitors treatment. At last, we analyzed the role of HDACs in the differentiation process of mammalian cochlea progenitor cells, especially differentiated into hair cells. This work not only makes progress in deciphering the role of epigenetic regulatory mechanisms in the development of PLL but also offers a greater understanding of epigenetic regulatory research towards intervention in human hearing defects.Part1Histone deacetylase activity is necessary for the migration of posterior lateral line primordium and deposition of neuromasts in zebrafish (danio rerio) larvaeObjectiveWe aim to investigate the histone deacetylases activity plays an indispensable role in posterior lateral line primordium migration and proneuromast deposition process.Materials and methodsAdding different concentrations of HDACs inhibitors to6hpf embryos, we observed the migration of posterior lateral line primordium and pattern of neuromasts by DAPI immunohistochemistry and cxcr7b whole mount in situ hybridization. We labeled proliferating primordium cells by addition of BrdU to the system water and observed the levels of acetylated histone H3and H4proteins by Western blot analysis of protein.Results1. VPA treatment caused concentration-dependent abnormal phenotypes and behavioral changes, which were described as lower hatching rate, curved body and the larvae were swimming in circles. High concentration of VPA (200μM) treatment caused the high level of cumulative lethality and the larvae did not reach the age of5dpf. We also found the increased levels of Ace-H3and Ace-H4in VPA and TSA-treated embryos by western blot analysis.2. We found the speed of primordium migration was significantly decreased under VPA and TSA treatment from6hpf comparing with the control embryos and this effect was dose-dependent Expression of cxcr7b in the developing PLL with whole mount in situ hybridization was also approved the effect of HDACs inhibitors on PLL primordium migration.3. We observed that HDACs inhibitors treatment altered the normal pattern of neuromasts, especially the LI position displaced posteriorly dependent on severity of the phenotype.4. We found a decrease in cell proliferation in the migrating primordia in HDACs inhibitors-treated morphants by analysis of immunohistochemistry.Conclusions1. Exposure to VPA leads to abnormal phenotypes and behavioral changes.2. HDACs inhibitors decreased the speed of PLL primordium (PLLP) migration.3. HDACs inhibitors have effects on the PLL neuromast spatial distribution and caused the position of neuromast drastically displaced posteriorly.4. HDACs inhibitors caused a decrease in cell proliferation within the primordium. Part2Histone deacetylase activity is required for hair cells differentiation within lateral-line-neuromast of zebrafishObjectiveWe aim to investigate the histone deacetylases activity plays an indispensable role in neuromast development in the lateral line system. HDACs activity is also required for the differentiation of hair cell.Materials and methodsWe added different concentrations of HDACs inhibitors (VPA (50,100,150μM), TSA (0.05,0.1,0.2μM) and MS-275(5,10,15μM)) to embryos (AB and Brm-GFP strains) from3dpf to5dpf or7dpf. We evaluated differentiated hair cells within neuromasts by myosin VI and GFP staining and labeled the functional hair cells by the mechanotransduction marker FM1-43FX. We labeled proliferating cells in neuromast by addition of BrdU to the system water and analysed apoptotic cells by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and cleaved caspase-3staining. We also observed the levels of AceH3, AceH4, AceH3K9, AceH3K14AceH4K5and AceH4K12by immunohistochemistry and western blot analysis of protein.Results1. Treatment with HDACs inhibitors decreased the number of hair cells within neuromast in a dose-dependent manner. The effect of HDACs inhibitors on the development of hair cells was also confirmed by the reduced FM1-43FX staining, which marks functional hair cells. We also found the increased levels of AceH3, AceH4, AceH3K9, AceH3K14AceH4K5and AceH4K12in HDACs inhibitors-treated embryos by immunohistochemistry and western blot analysis.2. We investigated the proliferation of lateral-line-neuromast cells and found that the inactivation of HDACs activity contributed to the decline in lateral-line-neuromast cells proliferation. Our data suggested that the treatment with VPA (1200μM) resulted in reduction in BrdU-labeled cells (VPA-treated8.1±0.3BrdU+cells per neuromast, n=35vs control11.8±0.4BrdU+cells per neuromast, n=46, p<0.05). Similarly, treatment of the neuromasts with TSA (0.1μM) and MS-275(10μM) also decreased the number of BrdU-labeled cells (TSA-treated8.2±0.3BrdU±cells per neuromast, n=35vs control11.6±0.3BrdU+cells per neuromast, n=45; p<0.05; MS-275-treated6.8±0.3BrdU+cells per neuromast, n=32vs control11.6±0.3BrdU+cells per neuromast, n=45, p<0.05).3. Under higher concentrations or long-time TSA treatment, such as0.2μM TSA treatment with4days, induced apoptosis as indicated by pyknotic nuclei in lateral-line-neuromast. The pyknotic nuclei were also TUNEL and cleaved-caspase3positive.Conclusions1. Histone deacetylation is essential for the development of the hair cells in the lateral-line-neuromast.2. HDAC inhibitors inhibited cell proliferation in the lateral-line-neuromast.3. Higher concentrations or long-time of TSA treatment induced cell apoptosis in the lateral-line-neuromast.Part3Histone deacetylase activity is necessary for the differentiation of cochlea progenitor cellsObjectiveWe aim to investigate the role of histone deacetylases activity in the process of P0mice cochlea progenitor cells differentiated into sensory epithelial cell, especially the hair cells.Materials and methodsAfter dissection of the cochlear tissues from ICR P0mice, these tissues were digested with trypsin, and stopped by the media containing10%FBS (fetal bovine serum). Then we collected the single cell, cultured in DMEM/F12media containing B27and N2supplements for7days. After7days’ suspension cultivation, cells formed spheres, and were collected for next differentiation period for another2weeks. The first week, we used the media containing TSA (0.1μM) or DMSO, respectively. We collected the cell spheres and the differentiated cells of the14th day for immunofluorescence and RT-PCR analysis. We examined the expression of Nestin、 BrdU、Pax2、myosin VIIA and Sox2by immunofluorescence analysis, and further detected the expression of genes, which are involed in inner ear development process by RT-PCR analysis.ResultsWe induced differentiation of the neonatal mouse cochlea progenitor cells with TSA treatment. And we found the number of newly formed myosinVIIA (+) hair cells and Sox2(+) supporting cells were decreased. We also found the number of both myosinVIIA (+) and BrdU (+) cells were decreased as well as the number of both Sox2(+) and BrdU (+) cells. RT-PCR results demonstrated the expression of hair cell markers as Mathl and myosinVIIA and supporting cell markers as p27kipl and Sox2were down-regulated throughout TSA treatment.Conclusions1. TSA treatment inhibited the differentiation of neonatal mouse cochlea progenitor cells into hair cells and supporting cells.2. TSA treatment inhibited the cochlea progenitor cells’ proliferation.