Tnfrsf11a-NF-κB Regulates The Formation Of The Lateral Line Canals By Activating Osteoclasts In Zebrafish

The lateral line system is a mechanosensory organ peculiarly exists in fish and amphibians,wnich is involved in the detection of surrounding water flow as well as the movement,speed and direction of living organisms.Consequently,it is implicated in many behaviors such as balance,tropism,feeding,predation avoidance,and communication.The lateral line system is composed of mechanoreceptors called neuromasts and their afferent neurons.The structure of the lateral line canals,such as the width of the canals and the degree of branching,determines the ability of the canal neuromasts to perceive water information.The morphology of lateral line canals is different among fish species,which is specie-specific and adaptable to the living environment and habits.At present,people have understood the formation process of lateral line canals,but the specific regulatory mechanism is not fully understood.The lateral line canals develop through a bone remodeling process from the scales or bones underlying the canal neuromast.Canal walls and roofs are associated with the activity of both osteoblasts and osteoclasts,indicating that osteoblasts and osteoclasts can participate in the formation of lateral line canals.Osteoclasts are bone resorptive cells derived from monocytes/macrophages of hematopoietic stem cells.RANK(receptor activator of NF-κB)encoded by tnfrsf11 a gene is a member of the tumor necrosis factor superfamily receptor,which plays an important role in bone remodeling.When RANK binds to its ligand(RANKL),the NF-κB signaling is activated,which turns on the transcription of downstream target genes and induces the differentiation of monocytes/macrophages-lineage cells into osteoclasts.OPG(osteoprotegerin)is encoded by tnfrsf11 b gene,which can competitively inhibit the binding of RANKL to RANK,thereby inhibiting the differentiation of precursor osteoclasts.Because of the role of NF-κB signaling in osteoclasts and bone remodeling,we investigated whether the NF-κB signaling affects the development of lateral line canals in fish.We first analyzed whether there is a connection between the NF-κB signaling and the lateral line canals.The results showed that NF-κB signaling reporter transgene was expressed on the trunk of zebrafish,while lateral line canals appeared after NF-κB signaling expression and co-localized with NF-κB signaling.The localization of osteoclasts further suggested that NF-κB signaling may be a factor affecting lateral line canals development.We constructed tnfrsf11 a mutant zebrafish by CRISPR/Cas9 knockout technology,and detected the base loss by sequencing and mRNA stability.At the DNA level,37 bases were inserted after the tnfrsf11 a gene was mutated,predicting premature termination of translation of the mutant protein.Moreover,the mRNA expression decreased significantly after the mutation,indicating that the tnfrsf11 a mutant zebrafish was successfully constructed.We found that NF-κB signaling located near the trunk lateral line canal was reduced and NF-κB target genes were downregulated in the mutant zebrafish.Subsequent analysis of osteoclasts in the lateral line scales of the mutant showed that the number and activity of osteoclasts were both significantly reduced.At the same time,the length and diameter of the lateral line canals and the number of hair cells within the canal neuromasts were decreased.These data demonstrated to us that tnfrsf11 a plays a role in the development of the lateral line canals in zebrafish,which is achieved by affecting NF-κB signaling and osteoclasts.tnfrsf11 a deficiency results in a significant reduction in activation of the NF-κB signaling pathway and therefore reduced osteoclast differentiation,resulting in an impact on the development of the final lateral line canal.In addition,the tnfsf11 a mutant zebrafish showed severe spinal curvature.We hypothesized that abnormalities in RANK and NF-κB may be a common cause of spinal anomalies and lateral line abnormities.Similarly,we also constructed tnfrsf11 b mutant zebrafish.At the DNA level,sequencing showed that the tnfrsf11 b gene was mutated with 36 bases insertion and 1 base deletion,which predicted that the translation of the mutant protein would terminate prematurely.After the mutation,the tnfrsf11 b mRNA expression was significantly decreased,indicating that the tnfrsf11 b mutant zebrafish was successfully constructed.However,no significant changes in NF-κB signaling were observed in the mutant zebrafish.The activity of osteoclasts was also not affected in the lateral line scales of the mutant,and the phenotype of the lateral line canal and the hair cells of the canal neuromasts did not change significantly.Therefore the mutation of tnfrsf11 b does not affect the development of the lateral line canals in zebrafish.We hypothesized that tnfrsf11 b may have speciesspecific effects on osteoclasts,or may only have different effects on osteoclasts in the zebrafish lateral line canals.It is also possible that the loss of tnfrsf11 b may lead to a compensatory effect of other factors to ensure the normal development of the zebrafish lateral line canals.In summary,we have found that Tnfrsf11 a expressed on the lateral line scales of zebrafish regulates osteoclast differentiation and bone remodeling by NF-κB signaling,which in turn is involved in the development of zebrafish lateral line canals.However,tnfrsf11 b mutation did not affect the development of lateral line canal in zebrafish.These works provide an important foundation for the study of the development and diversity formation of lateral line canals in fish.