닭 배아 망막에서 단일가닥 DNA 탐색자를 이용한 αB-crystallin mRNA 검출을 통한 성숙 희소돌기아교세포의 표지
Detection of αB-crystallin mRNA using Single-stranded DNA Probe in Oligodendrocytes of the Developing Chick Retina
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단일가닥 DNA 탐색자(single-stranded DNA probe)를 이용한 제자리부합법(in situ hybridization)은 탐색자 제작에 시간과 비용이 적게 들고 염색의 특이성과 민감도가 높기 때문에 특정세포의 전사물(transcript)을 관찰하는데 좋은 실험방법이다. 본 연구에서는 αB-crystallin (aBC) mRNA에 대한 단일가닥 DNA 탐색자를 제작하여 닭 배아의 발생 중인 망막에서 aBC mRNA 발현세포를 시공간적으로 관찰하였다. 역전사연쇄중합반응(reverse transcriptase-polymerase chain reaction)과 두 번의 연쇄중합반응을 통해 aBC mRNA에 대한 단일가닥 탐색자를 제작하여 제자리부합법으로 염색을 시행하였고 음성 대조를 위해 제작한 센스탐색자(sense probe)와의 비교 염색 및 aBC에 대한 면역조직화학 염색과의 비교를 통하여 단일가닥 DNA 탐색자의 높은 특이성을 확인하였다. 제자리부합법으로 염색한 발생 12일과 14일의 닭 배아 망막에서는 시각신경과인접한 부위에 존재하는 유두주위아교세포(peripapillary glial cell)에서 aBC mRNA 발현이 관찰되었고, 다른 부위에서는 양성반응이 관찰되지 않았다. 발생 16일에는 망막의 신경섬유층(nerve fiber layer)에서 적은 수의 aBC mRNA 발현세포가 관찰되었다. 발생 18일에는 망막의 신경섬유층과 신경절세포층(ganglion cell layer)에서 aBC mRNA 발현세포가 관찰되었고 발생 20일에는 동일한 부위에서 aBC mRNA 발현세포의 수가 증가한 것을 확인할 수 있었다. 발생 20일의 망막을 대상으로 aBC mRNA와 희소돌기아교세포(oligodendrocyte)의 표지자인nkx2.2의 발현 부위를 비교해 본 결과 분포 부위가 완전히 일치하였지만 신경세포의 표지자인 NeuN, 별아교세포의 표지자인 GFAP, 망막부챗살아교세포의 표지자인 vimentin 및 미세아교세포의 표지자인 RCA I과는 분포 형태에서 뚜렷한 차이를 보였다. aBC mRNA의 발현 시기와 세포 표지자와의 비교 염색을 통해 aBC mRNA 발현세포는 성숙 희소돌기아교세포임을 알 수 있었다. 본 연구는 닭 배아의 발생 중인 망막에서 aBC mRNA 발현세포를 시공간적으로 관찰한 최초의 보고이며, 향후 aBC mRNA에 대한 탐색자를 이용한 제자리부합법은 중추신경계통에서 성숙 희소돌기아교세포의 분포와 분화 연구에 유용하게 이용될 것으로 기대된다.
Abstract
In situ hybridization (ISH) using single-stranded DNA probe (ssDNA probe) is a useful method for observing the specific transcripts in cells, since it is convenient to prepare probe which is specific and sensitive. In this study, ssDNA probe for detection of αB-crystallin (aBC) mRNA, transcript of a heat shock protein, was prepared and aBC mRNA-expressed cells were spatiotemporally observed in the retina of the developing chick embryos. Single-stranded antisense probe produced by reverse transcription and polymerase chain reaction was identified as a specific probe for aBC mRNA in comparison to negative control using sense probe and immunohistochemistry for aBC protein. In the ISH experiment, aBC mRNA was expressed only in the peripapillary glial cells which are a specific cell type located in the avian retina adjacent to the optic nerve at E12 and E14 retinas. At E16, a small number of aBC mRNA-expressed cells were identified in the nerve fiber layer (NFL) of the retina. At E18, aBC mRNA-expressed cells were observed in the ganglion cell layer (GCL) as well as the NFL. At E20, the number of aBC mRNA-expressed cells was increased in the GCL and the NFL. Based on the same localization of nkx2.2immunoreactive cells and aBC mRNA-expressed cells, aBC mRNA-expressed cells were identified as oligodendrocytes. These results indicate that ssDNA probe for aBC mRNA detection is very useful tool for oligodendrocyte research such as distribution, migration and differentiation of the cells.
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