| Objective: Using in ovo electroporation,living tissue in vitro culture and immunofluorescence methods to investigate the role of the repulsive axon guidance protein,draxin,in the development of 23C10 positive neuronal axons in the chick hindbrain.Methods: Fertilized White Leghorn chick eggs were obtained from local commercial sources.Immunohistochemistry at HH18-19、HH21-22、HH25-26 chick hindbrains were used to observe the distribution and development of draxin and 23C10 positive neuronal axon.Draxin-AP fusion protein binding with living sections was used to check whether 23C10 positive neurons can bind draxin in vitro.Immunohistochemistry at HH25-26 chick hindbrain,which were overexpressed by the trans-membrane and secreted draxin plasmid at the stage of HH13-14,was used to observe the abnormal development of 23C10 positive neuronal axons.The function of draxin on the chick embryonic hindbrain neuronal axon growth was detected by in vitro culture method with the HH20-21 stage chick hindbrains.Results: After the overexpression of the trans-membrane form of draxin,23C10-positive axons were misprojected in the dorsal roof of the electroporated side of the hindbrain region.After the overexpression of secreted draxin,there were a few misprojected 23C10-positive axons in the dorsal roof of the electroporated side of the hindbrain region.There were no apparent 23C10-positive axons in the dorsal roof of the hindbrain region after the overexpression of the control vector and the normal group.Inhibition of axons outgrowth was found in the explants cultured by draxin conditioned medium,compared with the explants cultured in control conditioned medium.Conclusions:1 In the process of chick hindbrain development,the draxin protein is distributed around the 23C10-positive axons and the connection region of the 23C10-positive cranial nerves.The draxin protein distribution and 23C10-positive neuron and axon development have a temporal and spatial correlation.2 Draxin and 23C10 positive neurons and axons have the ability to combine with each other.3 From the levels of in vivo and in vitro,draxin has an inhibitory guiding effect on the projection of 23C10-positive neuronal axons in the chick hindbrain. |
