| BackgroundSonic Hedgehog (Shh) is a kind of secretory proteins, playing an important role in cell proliferation and differentiation, morphogenesis and axon guidance etc. In chicken embryonic spinal cord, Shh expresses in the notochord and floor plate and spread to form a strict concentration gradient controlling the formation of the dorsal-ventral axis. And by combining different receptors, Shh can attract or repel commissural neuron axon guidance, making the right connection between different neurons[3]. Studying the function of Shh helps us to better understand the mechanism of nervous system development.Objective1. To study whether there is any difference between different parts of the chicken embryonic spinal cord for the commissural neuron axons projection to rostral and caudal and whether the ratio of projection quantity for rostral and caudal is statistically significant.2. To study the effect of Shh overexpression to commissural neuron axon guidance during chicken embryonic development.3. To study the the expression of Pax3, Pax6, Pax7, N-Cdh, Cdh6 and Cdh7 in spinal cord during chicken embryonic development.4. To study the effect of Shh overexpression to Pax3, Pax6, Pax7 and Cdh7 in the spinal cord during chicken embryonic development.Methods1. At E3, green fluorescence protein (GFP) reporter gene plasmids pCAGGS-GFP was transfected into cervical, upper limb, rothorax, metathorax and posterior limb of the chicken embryonic spinal cord respectively by in vivo electroporation. At E6-E6.5, GFP positive embryos were collected and Open Book was used to observe the projection of nerve fibre clearly. Image J software was utilized to detect the gray-level of projection fibers, and then SPSS was used to to analyze Study whether there is any difference between different parts of the chicken embryonic spinal cord for the commissural neuron axons projection to rostral and caudal and whether the ratio of projection quantity for rostral and caudal is statistically significant.2. At E2.5-3, for control group, in vivo electroporation was used to transfect pCAGGS-GFP plasmid into one side of the chicken embryonic spinal cord. For experimental group, pCAGGS-Shh and pCAGGS-GFP plasmids were co-transfected by the same way. GFP positive samples were collected and sliced at E6. At least three samples were collected. Then the overexpression of Shh was detected using immunofluorescence histochemistry, and nerve fiber projecting affected by Shh overexpression was detected with Open Book technology.3. Spinal cords at E3-E6, E8 and E10 were collected during chicken embryonic development, then fixed, dehydrated, embedded with OCT and cut into frozen sections. The expression patterns of Pax3, Pax6 and Pax7 were detected by immunofluorescence at E4-E6. And N-Cdh, Cdh6 and Cdh7 were investigated with in situ hybridization in the spinal cord at different embryonic stages.4. At E2.5-3.0, for experimental group, pCAGGS-Shh and pCAGGS-GFP plasmids were co-transfected into one side of the chicken embryonic spinal cord by in vivo electroporation. Only GFP repoter gene plasmids were transfected by the same way as control group. Samples were collected and sliced at E4-E6. Then in situ hybridization was used to detect whether Shh was really overexpressed and the effect of Shh overexpression to Pax3, Pax6, Pax7 and Cdh7. Finally image J software and SPSS were utilized to detect the gray-level of two sides of the spinal cords and to analyze whether the expression quantity of Pax3, Pax6, Pax7and Cdh7 is statistically significant between the transfected side and untransfected side.Results1. For the five parts of embryo spinal cord, most commissural neuron axons project rostrally, while the minority of axons project caudally. For the ratio of projection quantity between rostral and caudal, five regions exist significant statistical difference between each other (P<0.01), except for the comparision between cervical and upper limb (P>0.05).2. The rsults of immunofluorescence histochemistry showed that Shh was overexpressed in the chicken embryonic spinal cord successfully. And compared with the control group, after Shh overexpression, the nerve fiber couldn’t across the floor plate and project stereotypically both in slice level and whole mount level.3. During E4-E6, the results of immunofluorescence histochemistry showed that Pax3 was mainly expressed in the alar plate, dermatome, dorsal root and dorsal root ganglion; Pax6 was mainly expressed in the middle of spinal cord across the alar plate and the basal plate; And the signal of Pax7 mainly existed in the alar plate, roof plate and dermatome. The results of in situ hybridization showed that, at the early stage, Cdh7 was mainly expressed in the dermatome, floor plate, dorsal domain of the basal plate, motor column, dorsal and ventral root. From E8 to E10, Cdh7 was mainly expressed in the gray matter and ventral root.4. The results of in situ hybridization showed that the overexpression model of Shh in chicken spinal cord was build successfully in the experimental group by in vivo electroporation. The results of immunofluorescence histochemistry and in situ hybridization showed that there existed highly significant difference between electroplated side and normal side (P<0.01). What’s more, the expression of Pax3, Pax6, Pax7 and Cdh7 were all depressed by Shh overexpression.Conclusion1. There exists difference for commissural neuron axons rostral projection and caudal projection among different regions of chicken spinal cord.2. Shh is a kind of axon guidance moleculars. Its overexpression can suppress the commissural neuron axon projection to the other side.3. Different expression patterns of Pax3, Pax6, Pax7, N-Cdh, Cdh6 and Cdh7 mean diverse roles for them in the spinal cord and its surrounding tissues during chicken embryonic development.4. The expression of Pax3, Pax6, Pax7 and Cdh7 in chicken spinal cord are all regulated by Shh signaling pathway. |
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