HIF Regulates Lateral Line Neuromast Development In Zebrafish Through Classical Wnt Signaling

The lateral line system is a unique sensory organ of fish and amphibians.Through the lateral line system,fish can sense changes in the surrounding water body and thus carry out behavioral activities such as feeding,avoiding enemies,flowing and migrating,which are of great importance to their survival.The neuromast is the basic structure that makes up the lateral line system,which is mainly made up of hair cells,supporting cells and mantle cells.The hair cells in the human inner ear are very similar to those in the zebrafish lateral line system but differ in terms of regeneration.The hair cells in the mature mammalian inner ear are not regenerated once damaged,whereas the hair cells in the zebrafish lateral line system can be regenerated.The development of the zebrafish lateral line system is therefore of great importance in the treatment of hearing impairment in humans.The HIF signaling pathway is a signaling pathway mediated by hypoxia-inducible factors（HIFs）that initiate the expression of downstream genes when cells are exposed to hypoxic conditions.A number of studies have shown that this signaling pathway is involved in the development of various organs and embryos,including blood vessels,kidneys and cartilage.However,little is known about the development of zebrafish lateral lineage hair cells.The present study focuses on whether the HIF signaling pathway is involved in zebrafish lateral line development.First,we used dimethyloxallyl glycine（DMOG）,a small molecule that inhibits prolyl hydroxylase domain-containing proteins（PHD）,to activate the HIF signalling pathway in zebrafish,and found that over-activation of The results showed that zebrafish showed not only some hypoxic responses,but also a significant decrease in the area of neuromast and the number of hair cells in the lateral line,tentatively concluding that over-activation of the HIF signaling pathway is detrimental to the lateral line development of zebrafish.In addition,we successfully constructed HIF signaling pathway-related gene mutant fish using CRISPR/Cas9 technology,including vhl^-/-mutants,hif1aa^-/-;hif1ab^-/-(abbreviated:hif1α^-/-)double mutants and epas1a^-/-;epas1b^-/-(abbreviated:epas1^-/-)double mutants.We examined the lateral line development of these three mutants using YO-PRO-1 and Sox2 to label hair cells and support cells,respectively,and found that both the 3 dpf and 5 dpf vhl^-/-mutants had significantly fewer hair cells and support cells than wild-type zebrafish.This result is consistent with the results obtained from DMOG treatment,suggesting that overactivation of the HIF signaling pathway is detrimental to the development of the lateral line in zebrafish.In addition,the number of hair cells in the lateral line of both hif1α^-/-and epas1^-/-double mutants was not significantly different from that of wild-type zebrafish.The q PCR assay revealed that the m RNA levels of epas1a were significantly upregulated in the hif1α^-/-double mutant,while the m RNA levels of hif1aa and hif1ab were significantly upregulated in the epas1^-/-double mutant,suggesting that the absence of changes in hair cells in the hif1α^-/-double mutant and epas1^-/-double mutant may be due to a reciprocal compensatory effect between members of the HIFs family.To further clarify whether vhl mutants and DMOG treatment affect the development of lateral line hair cells via Epas1 and Hif1αproteins,we treated vhl^-/-mutants using the HIF-1αinhibitor PX-478 and found that PX-478 partially rescued the reduction in vhl^-/-mutant hair cells.Meanwhile,we treated hif1α^-/-and epas1^-/-double mutants and wild-type fish using DMOG and found that epas1^-/-double mutant had significantly larger numbers of hair cells than wild-type zebrafish.The results suggest that the double mutations in epas1a and epas1b partially rescued the reduction in lateral line hair cells in zebrafish brought about by the DMOG-induced hypoxic response.To further investigate the reasons for the decrease in support cells and hair cells due to over-activation of the HIF signaling pathway,we used Brd U to label proliferating cells in 3 dpf to 5 dpf littermates,along with Sox2 antibody to label neuromast support cells,and identified proliferating support cells by co-localization labeling.The results showed that the number of proliferating support cells in vhl^-/-mutants was significantly less than the number of proliferating support cells in wild-type and heterozygous mutants(vhl^+/+and vhl^+/-).This suggests that activation of the HIF signaling pathway inhibits the proliferation of neuromast support cells.To investigate the specific mechanism by which the HIF signaling pathway affects hair cell development,we examined the expression of the Wnt signaling target gene（lef1）and the Wnt signaling downstream transcription factor Atoh1（including the atoh1a and atoh1b genes）in vhl^-/-mutants by q PCR and found that the m RNA levels of the lef1 and atoh1a genes were significantly reduced.We used the Wnt signaling transgenic reporter fish Tg（7x TCF-Xla.Sim:GFP）to explore whether over-activation of the HIF signaling pathway would have an effect on Wnt signaling.The results showed that when the HIF signaling pathway was activated using DMOG,the fluorescent signal on the neuromast of transgenic fish Tg（7x TCF-Xla.Sim:GFP）was significantly reduced.The results suggest that over-activation of the HIF signaling pathway causes a reduction in neuromast hair cells through inhibition of Wnt signaling.In summary,we found that activation of the HIF signaling pathway inhibits the development of zebrafish lateral line hair cells by suppressing Wnt signaling;when Epas1 and Hif1αproteins are inhibited,the loss of hair cells due to over-activation of the HIF pathway can be partially rescued.These works provide a basis for understanding the role of oxygen and hypoxic signaling in zebrafish lateral line hair cell development and provide theoretical support for the use of oxygen to promote subsequent hair cell protection and repair damage in mammals.