| The loss of mechanosensory hair cells owing to aging, genetic predisposition or environmental exposure to noise trauma or ototoxic drugs is permanent, and accounts for much of the hearing loss of human being. The sensory epithelium of birds and fish can regenerate new hair cells after deafening whereas humans and other mammals lost this ability during evolution. The strategy to treat sensorineural hearing loss is to manipulate the regeneration of human hair cells.The lateral line system is a sensory organ in fish and amphibians that detects water motion and pressure relative to the animal’s body. The lateral line comprises a series of individual neuromasts-cell clusters containing mechanosensory hair cells and surrounded by nonsensory support cells. Lateral line hair cells originated from placodes also share structural, functional and molecular similarities with the hair cells in the vertebrate inner ear. The lateral line system has become increasingly popular as a model for studying hair cell regeneration due to they are on the surface of the body and easily visualized after incubation of the fish with the vital fluorescent marker. It is important to investigate the capability and regular patten of hair cell regeneration in different stage of development, since there is a possibility effect of coming from development and self-renewal.Cell proliferation, apoptosis and differentiation procedure is involved in the patterning of mechanosensory organ (such as patterning of inner ear and development of neuromast), epigenetics may play a fundamental role in regulatory mechanisms controlling expression of certain gene. Histone code, covalent modification of histone tails especially histone lysine methylation is crucial for transcriptional regulation, mitotic chromosomal condensation, and heterochromatin formation.In eukaryocyte, methyltransferase and demethylase maintain the dynamic balance of histone methylation and demethylation. G9a is predominantly responsible for dimethylation of H3K9(H3K9me2). Previous study indicates that euchromatic H3-K9methylation regulated by G9a is essential for early embryogenesis and is involved in the transcriptional repression of developmental genes. Di-methyl lysine9on histone H3(H3K9me2) was noted in early differentiating retinal ganglion cells but was lost after birth.However, epigenetic control of inner ear development and hair cell regeneration is poorly understood. We use zebrafish lateral line as a model to study the roles of H3K9me2in hair cell patterning, development and regeneration.Part1The development of hair cells in the neuromast of zebrafishObjectiveWe aim to investigate the proliferation, apoptosis and differentiation of hair cells during the development of neuromast in zebrafish, which provided a model on illustrating the mechanism of hair cells generation.Materials and methodsThe wild-type AB zebrafishes of3-7days postfertilization (dpf) were employed. Functional mechanotransducing hair cells were labeled with the vital dye FM1-43FX, meanwhile rabbit anti-myosinVI immunofluorescence was also used to visualize lateral line hair cells. BrdU immunohistochemistry was used to detect cell proliferation. The in situ hybridization was performed to check the expression of fgfrl and atohla mRNA during the development of neuromast.Results1. It is the key stage for the development of hair cells in neuromast of zebrafish during3dpf to7dpf. Hair cells increased gradually after being hatched. The number of hair cells on different stage are presented by Mean±SEM, the difference between3dpf (4.100±0.2049, n=6) and4dpf (5.386±0.1809, n=14),4dpf and5dpf (8.467±0.3528, n=6) are significant, while there is no difference between5dpf and7dpf (9.225±0.4061,n=8)2. The cell proliferation is persistent during the development of neuromast. The nuclei of proliferative cells are labeled by anti-BrdU immunofluorescence, which is mainly situated in supporting cells along the periphery of neuromast, while seldom in hair cells. The difference on the cell proliferation between5dpf (2.300±0.3222, n=6) and7dpf (4.333±0.4244, n=6) is significant.3. The expression of fgfrl mRNA and atochla mRNA in the development of neuromasts. It was validated that FGF signaling pathway participated in the development of neuromasts by detecting the expression of fgfrl mRNA. Meanwhile, it has been accepted atohla plays roles during the differentiation of hair cells, in our research, it was validated there was atohla mRNA in the core of neuromasts on5dpf, while the expression decreased on7dpf, the difference was significant.Conclusions1. The neuromast on lateral line of zebrafish can be used as a model on investigating the proliferation and differentiation of hair cells.2. The whole mount in situ hybridization protocol on zebrafish neuromast was successfully established.3. The pattern of proliferation and differentiation of hair cells in neuromasts of zebrafish is recorded in detail, which will be helpful for the following research.Part2The regeneration of hair cells in neuromast of zebrafishObjectiveThe regeneration of hair cells in neuromast was investigated in different developmental stages of zebrafish, in order to establish the hair cells regeneration model and choose the optimized time window for neomycin treatment.Materials and methodsNeomycin treatments were performed on3dpf,5dpf and7dpf wild-type AB zebrafishes, then cell proliferation of neuroamst and regeneration of hair cells were observed at Oh,24h,48h and72h. Functional mechanotransducing hair cells were labeled with the vital dye FM1-43FX. BrdU immunohistochemistry was used to detect cell proliferation. The in situ hybridization was performed to check the expression of cmycb and odcl mRNA after neomycin treatment.Results 1. Apoptosis is the main cause of neomycin induced damage of hair cells in neuromast. Tinted Hair cells disappeared after treated by neomycin for1hour, meanwhile apoptotic bodies encased by red plasma membrane scattered in and around neuromasts.2. The regeneration of hair cells in different developmental stages of zebrafish. There were significant difference among3dpf(4.250±0.1794,n=12),5dpf(2.507±0.1433,n=15) and7dpf(1.333±0.1491,n=9) on the amount of hair cells at24hours post neomycin treatment. Meanwhile,24h,48h and72h post neomycin treatment on5dpf zebrafishes, hair cells of each neuromast increased gradually.3. Cell proliferation after the treatment of neomycin. The proliferative cells marked by anti-BrdU immunofluorescence increased after the treatment of neomycin, which was mainly situated in the nuclei of supporting cells, while was seldom observed in regenerated hair cells. The percentage of the number of double labeled hair cells in total hair cells is24.94±3.75%.4. The expression of c-mycb and odcl mRNA in neuromast after neomycin treatment. The regeneration was switched on after the treatment of neomycin, meanwhile the c-mycb and odc-1mRNA was detected in neuromasts.Conclusions1. The regeneration of hair cells on different developmental stage of zebrafish was examined systematically. The5dpf zebrafish is the optimized time window for the treatment of neomycin, because the continued differentiation of hair cells could be ruled out.2. The regenerated hair cells come from the transdifferentiation of supporting cells rather than proliferation.3. c-mycb and odcl take part in the regeneration of hair cells and apoptosis protection after neomycin treatment.Part3The role of H3K9me2on the development of neoromastsObjectiveThe expression of H3K9me2was investigated to explore the role on the development of neuromasts in zebrafish. Materials and methods1. The wild-type AB zebrafish of3-7dpf were employed. Functional mechanotransducing hair cells were labeled with the vital dye FM1-43FX, meanwhile mouse anti-H3K9me2immunofluorescence was also used to detect the expression of H3K9me2. DAPI was used to visualize the nuclei.2. The wild-type AB zebrafish treated by neomycin1h and underwent recovery for3days was employed. Functional mechanotransducing hair cells were labeled with the vital dye FM1-43FX, meanwhile mouse anti-H3K9me2immunofluorescence was also used to detect the expression of H3K9me2. DAPI was used to visualize the nuclei.ResultsThe expression of H3K9me2decreased during the development of neuromasts. The H3K9me2was strongly expressed in the neuromasts of3dpf zebrafish, while, decreased gradually along with the proceeding of hair cells differentiation, scattering at the outer proportion of neuromasts. The difference of the ratio of H3K9me2positive cells to hair cells between3dpf (1.830±0.09815, n=4) and4dpf(1.195±0.03175, n=4),4dpf and5dpf(1.060±0.1097, n=4),5dpf and7dpf(0.6250±0.04907, n=4) were significant.2. The expression of H3K9me2increased after the treatment of neomycin. Hair cells recovered after72h after neomycin treatment, while there was stronger expression of H3K9me2compared with control group. The difference on ratio of H3K9me2positive cells to hair cells between experimental group (1.153±0.07204, n=4) and control group(0.6250±0.04907, n=4) was significant.Conclusions1. H3K9me2takes roles on the development of neuromast and the regeneration of hair cells induced by neomycin.2. The expression of H3K9me2decreases along with the differentiation of haircells.3. The expression of H3K2me2is strong in the proliferating neuromast, while is weak in matured hair cells, which provides a hint that H3K9me2could be one of the key molecules in maintaining the characteristics of precursor cells in neuromasts. |
PDF Full Text Request