| Summary: | 碩士 === 國立陽明大學 === 神經科學研究所 === 88 === Abstract
Voltage-gated sodium channels (NaChs) are integral membrane proteins in the excitable cells. NaCh initiate and propagate action potentials when excitatory inputs achieve the threshold of depolarization. Multiple subtypes of sodium channels are expressed in the nervous system, which provide a valuable model for studying the regulatory mechanism of neuronal-specific genes. The success of transgenic approach in zebrafish prompts us to clone the upstream sequence of a zebrafish sodium channel gene and study its tissue-specific regulatory mechanism in vivo.
We have cloned a putative sodium channel gene expressing in the nervous system of zebrafish designed as SCN8A due to its sequence homology shared with rat SCN8A. SCN8A mRNA was detected in the trigeminal gangalion and Rohon-Beard neurons (RB) as early as 16 hpf (hour post fertilization) and at the dorso-lateral region of spinal cord since 24 hpf. We are interested in the temporal and spatial expression of SCN8A in the secondary motoneurons, because SCN8A mRNA was not expressed in the ventricular zone and barely expressed in secondary motor neurons which were located above the floor plate at 48 hpf. This study was focused on the expression of SCN8A at three differentiation stages including the proliferation at the ventricular zone, the migration and the final differentiation of secondary motoneurons at the spinal cord. We used antibodies against zHu (an early neuronal marker), DM-GRASP (a secondary motoneuron marker) and BrdU-incorporation (a mitotic marker) to correlate the expression of SCN8A mRNA in the spinal cord with the process of neural differentiation between 24 hpf and 72 hpf.
Our results indicated that both proliferation activity and differentiation of the spinal cord started from the anterior and extend into the posterior spinal cord. The majority of secondary motoneurons expressed low abundance of SCN8A mRNA as late as 72 hpf. At 5 days after fertilization, SCN8A mRNA expressing cells spread out the entire anterior spinal cord. In the posterior spinal cord, the medial and ventral region, SCN8A mRNA is still in low abundance. Several studies suggested that secondary motoneurons should be functional as early as 30 hpf. The delayed appearance of SCN8A mRNA in secondary motoneurons suggested that it was unlikely to play a major role during the initial episode of swimming unless a few of SCN8A-positive secondary motoneurons at the lateral edge of spinal cord is sufficient to trigger this motion.
The cell fates in the ventral central nervous system are induced by sonic hedgehog (shh), a member of the Hedgehog (Hh) gene family. Hh signaling is involved in the maturation of primary and secondary motoneurons. The ectopic expression of PKI can mimic the effects of overexpression of Hh family members. We were interested in the effect of ectopic expressions of Ihh and PKI on the expression of SCN8A mRNA in secondary motoneurons. We have reproduced phenotypes of the small-eye and excess of primary motoneurons in the injected embryo. However, the expression of SCN8A mRNA was not changed in the Ihh and PKI-injected embryos.
Several approaches were taken to clone the upstream sequence of SCN8A. Based on the sequences of 258 bp at 5￠ untranslated region (UTR) obtained by the previous cDNA library screening, we designed primers for 5￠ RACE and screened zebrafish genomic library twice to clone 5' upstream sequence of SCN8A. Fifty base pairs of further upstream sequence were obtained by 5' RACE. The sequence alignment of the first genomic clone with 5￠ UTR of SCN8A suggested that there was an intron located in the proximal to the translation start site. We designed primers based on this genomic sequence to screen the zebrafish BAC library. One candidate clones was obtained. Based on 5' UTR sequence, we initiated the inverse PCR and obtained 1313 bp fragment overlapping with 186 bp of 5' UTR. The fragment containing 1313 bp will be analyzed by the riboprobe protection to locate the transcription start site. The BAC clone and six clones obtained from the second screening of genomic library will be studied afterward.
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