The Collapsing Activity Of An Axon Guidance Cue Sema 3A And Its Expression In The Spinal Cord Of Chick Embryo

The normal development and correct projection of the central ne rv- ous system rely on the collaboration of various guidance cues in its growing environment. In recent years, these cues were identified as par- ticular axon growth attractants from the targets and axon growth repel- Ients from the nontargets. During the development of embryo, they are expressed around the growing axons. The cooperation of all these fic- tors induces the correct projection of different kinds of axons and then fabricates the complicated but coherent nervous net. Thus, they are de- fined as axon guidance cues. This study we detected the collapsing activity of an axon guidance cue Sema 3A to the growth cone of cultured NGF (nerve growth factor) sensitive neurons in DRG (dorsal root ganglia) from chick embryo. The expression of its mRNA in the spinal cord of different chick embryonic days was detected, which could be used to iluminate its guiding function and the mechanism of nervous net. Methods: 1. Methods to culture DRG explants from chick embryo were established. NGF sensitive neurons from dorsal root ganglia of chick embryos were cultured with reagent containing NGF. Sema 3A was added into the culture after 18-24h of culture. The morphologic changes of growth cones were observed and photographed. 2. Plasmid containing Sema 3A encoding sequence was amplified and RNA probe was produced. The expression of Sema 3A in the spinal cord of chck embryo was detected by using in situ hybridization. -3- Results: 1. After 1 8-24h culture, many axons grew outwards with growth cones at the terminal. There were lamellipodias and filopodias spreading and waving on the growth cone, After Sema 3A was added, the spreading and waving stopped and the growth cones retracted like dry sticks. That is, the growth cones collapsed. 2. In the 4th embryonic day (E4), sema 3A mRNA was expressed around the neural tube and at the notocord. In 136 and E7, it was expressed in the ventral horn and around the central tube, appearing ventral dominance. To E8 embryonic day, the expression was mainly in the ventral horn, especially in the motor neurons. The acute reaction of neurons against axon repellents is the collapse of growth cones, while the chronical reaction is the change of axon growth direction when they are cocultured with repellents secreting cells. In our study, sema 3A could induce the collapse of the growth cone of those NGF sensitive neurons in DRG proving that sema 3A is the guidance cue of these neurons. From 134 to 136 of chick embryo, sema 3A mRNA is expressed around the neural tube, the notocord and the ventral horn. This contributes to preventing sensoiy axons of DRG from entering the gray matter in waiting period. From 136 to 138, the expres- sion retrieved ventrally and expressed at the ventral half of the central tube (137) and the ventral horn especially in motor neurons (E7 and E8). Thus, the dorsal terminating axons relating to nociception and thermore? ception can enter the dorsal-most laminae I and II but not grow further ventrally. As for the waiting period and the correct projection of the NT- 3 (neurotrophin-3)sensitive Ia neurons and other neurons, it is possible that other guidance cues might make more contribution.