Effect of sonic hedgehog on motor neuron positioning in the spinal cord during chicken embryonic development

Abstract Sonic hedgehog (SHH) is a vertebrate homologue of the secreted Drosophila protein hedgehog and is expressed by the notochord and floor plate in the developing spinal cord. Sonic hedgehog provides signals relevant for positional information, cell proliferation and possibly cell survival, depending on the time and location of expression. Although the role of SHH in providing positional information in the neural tube has been experimentally proven, the underlying mechanism remains unclear. In this study, in ovo electroporation was employed in the chicken spinal cord during chicken embryo development. Electroporation was conducted at stage 17 (E2.5), after electroporation the embryos were continued incubating to stage 28 (E6) for sampling, tissue fixation with 4% paraformaldehyde and frozen sectioning. Sonic hedgehog and related protein expressions were detected by in situ hybridization and fluorescence immunohistochemistry and the results were analysed after microphotography. Our results indicate that the ectopic expression of SHH leads to ventralization in the spinal cord during chicken embryonic development by inducing abnormalities in the structure of the motor column and motor neuron integration. In addition, ectopic SHH expression inhibits the expression of dorsal transcription factors and commissural axon projections. The correct location of SHH expression is vital to the formation of the motor column. Ectopic expression of SHH in the spinal cord not only affects the positioning of motor neurons, but also induces abnormalities in the structure of the motor column. It leads to ventralization in the spinal cord, resulting in the formation of more ventral neurons forming during neuronal formation.

lead to important defects of in MN positioning within the spinal cord during embryonic development. 10,11 Although the role of these transcription factors in MN positioning in the spinal cord is well established, little is known regarding their potential effector genes. 6 Sonic hedgehog (SHH) is a vertebrate homologue of the secreted protein encoded by the Drosophila gene hedgehog, 15,16 and is expressed by the notochord and floor plate at the time when these structures exert their inductive activities. 17,18 In the central nervous system, SHH plays an important role in the ventral specification along the entire neural axes. In ventral regions, this protein acts as a long-range graded signal that controls the pattern of neurogenesis. 19,20 Misexpression of SHH in vertebrate embryos can induce the differentiation of floor plate cells at ectopic locations in the neural tube. 18,21,22 Sonic hedgehog provides signals relevant to positional information, cell proliferation, and possibly cell survival depending on the timing and location of the expression. 17,23,24 Although the role of SHH in providing positional information in the neural tube has been experimentally established, the mechanism underlying this phenomenon remains unclear.
In this study, we focus on the role of SHH in motor neuron positioning in the spinal cords during chicken embryonic development by inducing its misexpression. We examined the gene ex-

| In ovo electroporation
Sonic hedgehog plasmid was gifted by Redies (Prof. Dr Christoph Redies, Institute of Anatomy I, Jena University Hospital, Teichgraben 7, D-07743 Jena, Germany). pCAGGS-GFP (green fluorescent protein) plasmid was constructed by our own lab. All plasmids used in this research were extracted by a plasmid extraction kit following the manufacturer instructions (Cwbio, Beijing, China).
The method of in ovo electroporation was performed as described previously with a few modifications. 24,26,27 Briefly, after 2.5 days incubation (E2.5), fertilized eggs were transferred from the incubator to clean benches, and where 3-4 mL albumin was carefully removed without disrupting the yolk. Then, a 2-3 cm diameter window on the shell was opened by scissors without hurting the embryo. pCAGGS-SHH (4 µg/µL), pCAGGS-GFP (0.25 µg/µL) plasmids and Fast Green dye (0.01%) were mixed together as a working solution for the ectopic expression group. Solution without pCAGGS-SHH was used in the control group. Plasmids were injected into the neural tube lumen using a capillary needle under a stereomicroscope. Electrodes were then immediately placed parallel to each other on both sides of the spinal cord. The electroporation parameters were volt 18 V, six times pulses, 60 ms/pulse and a 100 ms interval (CUY-21, Nepa Gene, Ichikawa, Japan). Bubbles around the positive pole indicated successful electroporation. After completing electroporation, eggs were sealed with ventilated tape and replaced into an incubator for continuous development. Sample collection and analysis were performed at the desired stage. For newborn neuron tracing, 5 μg/μL bromodeoxyuridine (BrdU) was added into the embryo keeping for 24 hours before sample collection.

| Tissue sectioning
The spinal cords of E6 (stage 28) embryos were collected and immersed in 4% paraformaldehyde (PFA) solution for 6-24 hours according to tissue size. Then 18% sucrose solution was used to replace PFA and dehydration. The spinal cord was embedded in OCT compound (Sakura Finetek Torrance, CA, USA) and stored at −80°C.

| Microscopy
The chicken embryo was imaged by a stereo fluorescence microscope (LEICA M205FA; Leica Microsystems CMS GmbH, Wetzlar, Germany), which was equipped with a digital camera (LEICA DFC425C; Leica Microsystems CMS GmbH, Wetzlar, Germany). A confocal microscope (Olympus ix81; Olympus, Kyoto, Japan) was used to observe immunofluorescence sections. A microscope (Nikon ECLIPSE 80i; Nikon, Tokyo, Japan) equipped with a digital camera (LEICA DFC300FX; Leica Microsystems CMS GmbH, Wetzlar, Germany) was used to observe other cryosections without fluorescence.

| Data analysis
Image-Pro 6 version software was used to measure the optical and fluorescence intensity of captured images (Media Cybernetics, Rockville, MD). There were 3-5 independent experiments for each group and all data were presented as the mean ± SD. Data in different

| SHH ectopic expression in the developing chicken spinal cord
In ovo electroporation, a technique by which a plasmid can be uni-  Figure 4U-X), and the ratio of the transfected to non-transfected sides was 0.53 ± 0.27 (n = 3). In the control group, no differences were observed ( Figure 4Z-C'), and the ratio was 1.17 ± 0.11 (n = 3). As shown in Figure 4Y, the ratio of the number of BrdU-positive cells on the transfected side to that on the non-transfected was significantly different in the SHH ectopic expression group compared to control one (P < 0.01, Figure 4D').
Interestingly, at stage 24 (E4), SHH promoted neuroepithelial cell proliferation ( Figure 4J), while at stage 28 (E6) it did not affect cell proliferation ( Figure 4T, D'). Therefore, we speculated that SHH not only affects the proliferation of neural precursor cells but also their differentiation.

| The effect of SHH ectopic expression on MAP-2, neurofilament and the growth of commissural axons
Interestingly, MAP-2 labelling of motor columns following SHH ectopic expression in the spinal cord revealed structural abnormalities ( Figure 6A-H, the arrow is shown). In the control group, the structure of the MAP-2 labelled motor column was normal ( Figure 6I-P).
Motor neurons express MAP-2, even though MAP-2 is not a specific    38 Our results showed that the transfected side of the spinal cord as compared to the non-transfected side has obvious differences in

| CON CLUS ION
The