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ゼブラフィッシュにおける下オリーブ核ニューロン分化の分子機構の解明

伊藤, 翼 名古屋大学

2021.06.23

概要

小脳は運動制御や運動学習に関与している器官であり、その神経回路は哺乳類からゼブラフィッシュのような真骨魚類まで広く保存されている。小脳神経回路は小脳外から2つの入力を受けている。1つは小脳前核から伸びる苔状線維で、もう1つは下オリーブ核から伸びる登上線維である。苔状線維は顆粒細胞を介し、プルキンエ細胞に情報を伝達し、登上線維はプルキンエ細胞に直接投射し、情報を伝達している。これらの情報はプルキンエ細胞で統合され、小脳外に出力し、小脳は機能を果たしている。これらの小脳ニューロンは発生の初期段階で後脳領域背側に位置する神経前駆細胞から産生される。この後脳領域は菱脳節と呼ばれる第1-7までの分節構造を持っている。特に、後脳後方(第4-7菱脳節)のパターン形成には第4菱脳節で産生されるFgf3/8aと体節で産生されるレチノイン酸の濃度勾配が関与していることが知られている。マウスでは後脳領域で発現するプロニューラル遺伝子であるPtf1a(ゼブラフィッシュの場合はptf1a)から様々なニューロンが分化する。第1菱脳節のPtf1a陽性細胞からはプルキンエ細胞、第2-5菱脳節からは蝸牛神経核ニューロン、第7菱脳節からは下オリーブ核ニューロンが分化する。しかしながら、ptf1a陽性神経前駆細胞からどのようにしてこのような様々なタイプのニューロンが分化するのか、どのように運命決定が行われているのかは明らかになっていない。

本研究において、トランスジェニック(Tg)系統を用いた解析から、ホメオボックス遺伝子であるgsx2が後脳後方の背側領域の下オリーブ核ニューロン前駆細胞で発現し、ptf1aと共発現していることが明らかとなった。そこでさらなる解析を行うためCRISPR/Cas9法を用いてgsx2とptf1aの変異体の作製を行い、表現型解析を行った。その結果、受精後5日目の仔魚において、gsx2、ptf1a変異体では下オリーブ核ニューロンが著しく減少、あるいは完全に消失していることが明らかとなった。さらに、ptf1a変異体とgsx2変異体を用いて、発現解析を行ったところ、gsx2とptf1aが遺伝子のカスケードの上下関係にないことが明らかとなった。このことから、gsx2とptf1aが下オリーブ核ニューロンの形成において、それぞれ独立して機能することが明らかとなった。

また、化学阻害剤やアンチセンスモルフォリノオリゴヌクレオチド(MO)を用いて、レチノイン酸やFgf3/8aのシグナルを阻害し、gsx2の発現や下オリーブ核ニューロンの形成への影響について解析を行った。その結果、レチノイン酸はgsx2の発現を正に制御しており、Fgf3/8aシグナルはgsx2の発現を負に制御していることが明らかとなった。さらに、第5-6菱脳節で発現するmafbaの変異体を用いた解析、第7菱脳節で発現するhoxb4aの過剰発現による解析、pbx2/4MOを用いた発現阻害の解析結果から、レチノイン酸やFgf3/8aのシグナルの下流で、mafba、hoxb4aなどのHox遺伝子が発現し、Hox遺伝子に関してはPbx2/4と複合体を形成することで、gsx2の発現を制御し、ptf1a陽性神経前駆細胞から下オリーブ核ニューロンへの運命決定を担っていると考えられる。

今後は、Gsx2やPtf1aが発現を制御する遺伝子の存在を明らかにしていくことで、下オリーブ核ニューロンの分化の分子機構の全容が明らかになることが期待される。

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