Zebrafish Gfi1.2 Gene Activity In The Inner Ear

Deafness effects the health of human seriously.Hearing loss has a high incidence-about 278 million people suffer from moderate to severe hearing loss in the world. Incidence reduction, early detection, and early treatment of hearing loss have become a common interest of society. Prevention and treatment of sensorineural deafness basic research is still hot and difficult.The prevention and treatment of sensorineural deafness is a central and difficult issue in the basic science research field. One of the frontiers of research is to study the gene function and the molecular mechanisms of deafness. By knocking out or importing genes in ways using animal models improve the study of gene function in deafness.These genes play important roles in the development of hair cell, regulation of transcription factors and et al. All of them control the development of the inner ear from the perspective of spatial or time distribution through co-ordination.In recent years, with the development of techniques in molecular and cellular biology, many discoveries of new genes and specific phenotype related to inner ear development have been made.Growth factor independence-1(Gfil) encodes a transcription repressor factor which plays important biological roles in hematopoietic, neuron and inner ear hair cell development. It determines cell fate determination and differentiation by cell apoptosis and proliferation. It is mainly expressed in the mouse hair cells and neurons of inner ear. In Gfil-deficient mice, the vestibular and cochlea hair cells are differentiated but with abnormal morphology and the related nerve distributions are also abnormal. So it plays a crucial role in differentiation and survival of hair cell.However, nothing is known about whether Gfil gene is required for the normal development of vertebrates. To further study the regulation of gfil in zebrafish inner ear hair cells and neurons, we used antisense morpholino oligonucleotides (MOs) to knockdown and overexpress certain genes.Zebrafish has two homologous genes gfil-1 and gfil.2. First we investigated the expression patterns and sequenced characteristics of gfil.1 and gfil.2. Comparison of the vertebrate proteins revealed that the zebrafish gfil.2 protein is more homologous to Gfil than to Gfilb.Transcripts of both genes were present in fertilized eggs, suggesting a maternal origin.They all expressed mainly in neuraltube of ectoderm in gastrula stage.From early segmentation 19hpf they both played a role in the development of inner ear hair cells and neural tissue of brain. However, gfil.2 was expressed even earlier in the inner ear at 14hpf and was essential during the differentiation of the precursor cells in the inner ear.To understand the relationship between the two genes and the role in the developing inner ear, by knocking down gfil.1 or gfil.2 and both changed the expression of ngnl, neurod and isletl.Underthe same condition, MO injection did not affect markers involved in pre-placodal ectoderm and otic placode and differentiation of hair cell. We further discovered that knocking down one or both of gfil.1 and gfil.2 caused growth delay and reduction of the number of hair cells and the neurons.The study shows that the lack of gfil.1 and gfil.2 will affect the development of hair cells and neurons. Both of them have redundant function, if one single gene is suppressed then the other homologous gene for functional replacement.Due to gfil.2 playing an early role in inner ear development, this study further explored gfil.2 gene in the early formation of inner ear sensorineural cells. The results showed that during the 11hpf of zebrafish embryonic development, over expression of gfil.2 promoted the expression of ngnl, atohlb and could rescue the effect of gfil.2 MO. It shows that the effect of gfil.2 on ngnl and atohlb is specific. To further explore the mechanism of regulation on ngnl, over-expression id2a can decrease ngnl expression, while overexpression gfil.2 and id2a can save the effect on ngnl. These results suggest that by inhibiting the id2a, gfil.2 promote the early expression of ngnl.In summary, gfil.2 may regulate proneural genes ngnl and atohlb promoting early sensoryneural progenitor cell differentiation,which ultimately affect the hair cells and the number of neurons.This is the first study exploring the function of gfil.2 in the zebrafish inner ear.It provide direction to study the function of gfil.2 in future research of neural precursor cells just stem cells.