Wls在斑馬魚胚胎內對原神經基因表現所扮演的角色

Abstract

作為演化上高度保留的訊息傳遞路徑，Wnt參與了早期胚胎發育過程中的許多調控。而Wntless，對於Wnt蛋白的分泌扮演了非常重要的角色。neurogenin-1 (ngn1)是一種轉錄因子，主要表現在早期神經系統中，神經前驅細胞的區域(Progenitor zone)。作為一種神經前驅型基因(Proneural gene)，ngn1參與了早期多功能幹細胞至神經細胞的分化過程。在過去的研究中發現，在Wntless突變的斑馬魚的間腦(Diencephalon)，ngn1的表現量大幅度下降。而ngn1的表現量下降，很可能干擾了斑馬魚胚胎的缰核(Habenula)神經元的形成。然而，於此過程中，wls與ngn1間的調控機制目前尚不清楚。 在此，我們藉由螢光原位雜合技術 (Fluorescent in situ Hybridization)、免疫染色(Immunostaining)與3-D影像對位(3-Dimensional Image Registration)的技術，來評估在wntless突變胚胎中ngn1與her6 (一個ngn1的抑制子)之間的表現情形。並且藉由細胞譜系標記(Cell Lineage Labeling)的方式，分析ngn1啟動子驅動的細胞與韁核之間的關係。過程中發現，於間腦處的ngn1的表現量減少了近40%，而her6的表現量增加近4.5倍。而藉由分析了ngn1與her6之間的消長與空間位置的關係，顯示出在神經細胞分化的過程中，可能出現了細胞命運轉換(Cell Fate alteration)的情形。除此之外，我們的結果顯示，細胞命運轉換的現象很有可能是影響缰核發育的重要原因。

Wnt signaling pathway is a conserved pathway regulating critical evens during embryonic development in different animals. Wls plays an important role in the secretion of Wnt protein. Neurogenin 1 (ngn1) is a transcriptional factor that is mainly expressed in the progenitor zone in the neural system. As a proneural gene, ngn1 derives the differentiation of pluripotent stem cells to neuronal subtypes. In the previous study, reduction of ngn1 expression in zebrafish wls mutant is proposed to perturb habenular neuronal development in the zebrafish embryos. However, the regulatory relationship between ngn1 and wls is still unclear. Utilizing fluorescent in situ hybridization (FISH), immunostaining and 3-dimensional image registration techniques, the expressions of ngn1 and her6, a repressor of ngn1, in zebrafish wildtype and wls mutant embryos have been evaluated. The results indicate that ngn1 expression exhibits nearly 40% reduction whereas her6 expression exhibits 4.5 times increase in the HA progenitor zone in diencephalon of the zebrafish wls mutant embryos. The increased her6 and reduced ngn1 expressions combined with their positional relationship suggest that the alteration of cell fate occurs between the generations of diencephalic her6 and ngn1 proneurons. Our results suggest that the alteration of cell fate conceivably perturbs the development of habenular nuclei.