Sonic Hedgehog Regulates The Pathfinding Of Descending Serotonergic Axons In Hindbrain With Wnt5a And Secreted Frizzled-Related Protein 1 Collaborations

Objective:Sonic Hedgehog is one of the key molecules that define serotonergic neurons fate specification and regulate axons pathfinding in spinal cord.Previous study has proved Wnt/PCP signaling pathway attracts serotonergic axons pathfinding in brain stem.Collaborations of Sonic Hedgehog and Wnt signaling pathways have been shown to play an important role during commissural axons projection along the longitudinal axis toward the brain,and collaborations of them are modulated by Sfrp1.Sfrp has been proven to be a transcriptional target of Shh in developmental neural tube.Meanwhile,Sfrp1 acts as an inhibitor of canonical and noncanonical Wnt signaling pathways.In order to explore whether Shh and Wnt/PCP(Wnt planar cell polarity)signaling pathways collaborate to guide descending 5-HT axons pathfinding via Sfrp1,we performed in utero electroporation and injection,and Dunn chamber assay with cultured descending 5-HT neurons.Method:We analyzed the expression of Shh signaling pathway components Patched1(Ptch1)and Smoothened(Smo),and Wnt/PCP signaling pathway components Frizzled3(Fzd3)and Vangl2 in descending 5-HT neurons of E12.5 mouse hindbrain by immunostaining.Next,using immunostaining and western blotting we analyzed the expression pattern of Shh,Sfrp1 and Wnt5a in E12.5 posterior hindbrain.To investigate the collaborations of these proteins to guide descending 5-HT axons pathfinding,E12.5 cultured descending 5-HT neurons were treated with different gradients,respectively,including BSA gradient,Sfrp1 gradient,Wnt5a gradient,Shh gradient,Wnt5a and Sfrp1 gradient(the same directions and opposite directions),Wnt5a and Shh gradient,Shh and Sfrp1 gradient.Next we performed in vivo electroporation of pIRES-EGFP or pIRES-Shh-EGFP on E12.5 mouse embryos hindbrain to examine whether Shh overexpression could disrupt 5-HT axons descending.Using biochemical methods we tested whether Shh overexpression could upregulate Sfrp1 and interrupt Wnt5a binding to Fzd3 in E12.5 hindbrain.Mouse E12.5 embryos were injected with Sfrpl protein or co-injected with Sfrp1 protein and WAY-316606,and DMSO was used as control.Embryos were dissected out at E14.5 to detect descending 5-HT axons pathfinding.Result:The expression of Shh signaling pathway components,Smo,Ptchl and Wnt/PCP signaling pathway components,Fzd3,Vangl2 was detected in descending 5-HT neurons somas and growth cones.In E12.5 mouse brain stem,we found both Shh and Sfrp1 were expressed higher at R4(rhombomere 4)and gradually decreased along the posterior direction.In contrast,Wnt5a expression was lower at R4 and gradually increased along the posterior direction.Moreover,we reached basically the same conclusions on Western blotting and immunostaining.E12.5 cultured descending 5-HT neurons showed no response to BSA or Sfrp1 gradient.Treated with low to high Wnt5a gradient,growth cones turned to the higher concentration of Wnt5a.However,growth cones turning dramatically reduced when co-treated with low to high Wnt5a and Sfrp1 gradients.In contrast,co-treated with low to high Wnt5a and high to low Sfrp1 gradients,growth cones turned dramatically increased.Growth cones turned to the lower concentration of Shh when treated with high to low Shh gradient.However,co-treated with high to low Shh and low to high Wnt5a gradients,growth cones turned remarkably increased comparing to treat with high to low Shh or low to high Wnt5a gradient.Moreover,there was no dramatic difference in growth cones turning after co-treating with high to low Shh and Sfrp1 gradients,comparing to treat with high to low Shh gradient.Shh overexpression disrupted 5-HT axons descending.Furthermore,biochemical results showed Shh overexpression upregulated Sfip1,but had no detectable effect on the expression of wnt5a;Shh overexpression led to increase of Sfrp1-Fzd3 binding rate and interrupted Wnt5a binding to Fzd3.Sfrp1 overexpression resulted in pathfinding error of descending 5-HT axons.Nevertheless,co-injected with Sfrpl and Sfrp1 antagonist WAY-316606,most of the axons turned normally along the A-P axis.Conclusion:Shh and Wnt5a collaborated to guide descending 5-HT axons pathfinding.Shh overexpression disrupted 5-HT axons descending via upregulating Sfrp1 and interrupting Wnt5a binding to Fzd3.Therefore,our results indicated that Shh,Sfrp1 and Wnt5a collaborated in guiding descending 5-HT axons projection.