Shh-Ptch1-Smo Signaling Pathway Regulates The Guidance Of Ascending Serotoninergic Axon

The serotoninergic nervous system is one of the most important nervous system in the central nervous system.The 5-HT axons are originated from serontoninergic neurons in raphe nuclei that produce 5-HT neurotransmiter.The serotoninergic nervous system is involved in many important neural functions,including sexual orientation,analgesia and emotional control.However the pathfinding of the distinct axonal projections of serontoninergic axons still is not understand exactly.Our previous study has proved that Shh-Ptch1-Smo signaling guides spatial pathfinding of descending 5-HT axons in the spinal cord by multidirectional repulsion.But the molecular mechanism underlying Shh regulating the pathfinding of ascending 5-HT axons had remained unclear.Here we present bidirectional expression gradients of Shh in the embroynic midbrain.In addition,we also provide in vitro,ex vivo and in vivo evidence that the Shh-Ptch1-Smo signal attracts the ascending 5-HT axons in the midbrain of embryonic mice.Objective: To study mechanism underlying Shh regulating the axon guidance of ascending 5-HT axons.Methods:(1)We analyzed the gradient of Shh mRNA in E12 mouse hindbrain by using in situ hybridization.Western blotting was performed to examine the gradient of Shh protein in the E12 hindbrain.(2)Using immunostaining on tissue sections and cultured neurons,the expression of Shh signaling pathway core components Ptch1 and Smo was analyzed in E11 and E12 ascending 5-HT neurons to assess whether Shh signaling regulates the pathfinding of ascending 5-HT axons.(3)A Dunn chamber assay was established to verify that Shh protein attracts ascending 5-HT axon projections.(4)We turned to ex vivo and in vivo analyses to further verify the roles of Shh signaling and its downstream molecules Ptch1 and Smo in ascending and descending 5-HT axon guidance.Results:(1)Shh expression is lower at rhombomere 4(R4)and gradually increased in the posterior-to-anterior(P-A)direction(R4 low to midbrain high).(2)Ascending 5-HT neurons expressed core Shh signaling components Ptch1 and Smo.(3)Shh protein attracts ascending 5-HT axons through Ptch1 and Smo.(4)Ptch1 and Smo are required in the Shh guidance of 5-HT axons.Conclusions: 1.Shh attracts ascending 5-HT axons through Ptch1 and Smo.2.Shh is a bidirectional axon guidance cue during the pathfinding of 5-HT axons along the A-P direction.