利用果蠅遺傳學探討ARMS在神經系統發育中的功能

Abstract

ARMS (Ankyrin repeat-rich membrane spanning)，也稱 Kindins220 (kinase D-interacting substrate of 220 kDa)，是在演化上從線蟲到人類都有的高度保留基因。ARMS可以在許多組織中被偵測到，尤其最大量表現在發育中或成體的神經組織中，暗示其在神經系統中的重要性。之前的研究指出ARMS是酪胺酸激酶受體的下游分子，將訊號傳遞經由有絲分裂原激活蛋白激酶途徑(mitogen-activated protein kinase, MAPK)來引發有絲分裂原激活蛋白激酶持續的磷酸化，進而調控神經元的極性與神經纖維的生長。 此篇我們利用果蠅遺傳學來研究ARMS在神經系統中所扮演的角色。先前實驗室已發現在幼蟲時期,將眼碟經由核醣核酸干擾(RNA interference)使其ARMS減少，會造成成蟲複眼缺陷。此結果驅使我們更進一步去細究是否那些缺陷是發生在更早的發育時期。在檢視三齡幼蟲時期之果蠅視葉(optic lobe)的光受器1到8 (R1-R8)之後，並未發現任何缺陷。我們也檢查在三齡幼蟲晚期的其他神經器官，包括蕈狀體 (嗅覺學習與記憶中心)、腹部神經索(用來處理與傳送感覺神經傳來的訊號以及將訊號傳遞給運動神經)和周邊神經，觀察ARMS減少時是否會對其造成影響。結果顯示蕈狀體和腹部神經索沒有被ARMS減少所影響。然而，我們發現三齡幼蟲晚期，其周邊神經系統中的第四級樹突分枝神經元的ddaC，和控制組比較下，ARMS減少時呈現樹突複雜度降低。Sholl氏分析和Neurolucida分析證實ARMS減少所造成的樹突複雜度降低是由於其較高級數的樹突神經纖維分枝減少、樹突末端點的數量也減少。但是ARMS減少並不影響從神經細胞本體所長出之第一級樹突的數量與細胞本體大小。為了更進一步了解ddaC樹突複雜度降低是因為樹突發育不良還是維持不良，我們觀察了更早時期，包括二齡幼蟲中期和三齡幼蟲早至中期的ddaC。此外，我們證實在ddaC中所見到的缺陷並不是因為核醣核酸干擾失準，因為過度表現ARMS可以救回ARMS減少所造成的缺陷。 已知ARMS是ephrin/Eph受體的下游分子，包含在有絲分裂原激活蛋白激酶途徑中,也有可能包含在磷脂醯肌醇激酶(phosphoinositide 3-kinases; PI3K)途徑裡，因此我們在有ARMS被核醣核酸干擾的背景下，與變異的Eph、PI3K或 MAPK等個別做結合表現，檢視其對三齡幼蟲晚期的ddaC之樹突複雜度的影響。

ARMS (Ankyrin repeat-rich membrane spanning), also known as Kindins220 (kinase D-interacting substrate of 220 kDa), is an evolutionarily highly conserved gene from nematode to human. ARMS can be detected in several tissues, but is most abundantly expressed in the developing and adult neural tissues, suggesting its biological role in the nervous system. Previous studies demonstrate that ARMS is a downstream target of receptor tyrosine kinase (RTK), which signals via MAPK (mitogen-activated protein kinase) pathway for sustained MAPK phosphorylation. By this way, ARMS regulates neuronal polarity and neurite outgrowth. To investigate ARMS role in the nervous system, we have exploited Drosophila genetics. RNAi mediated knockdown of ARMS with driven expression in the eye disc in larvae stage results in defect of drosophila ommatidia in adult fly. This finding prompts us to further dissect whether such defect occurs in earlier developmental stage. By examing photoreceptors R1-R8 of optic lobe at late-third larval stage, we do not observe any defect. We thus turn to screen mushroom body (organ for olfaction and memory storage), ventral nerve cord (for transducing and processing signaling from sensory neurons to motor neurons), and the PNS (peripheral nervous system) in the developing nervous system at late-third instar stage to search for neuronal defect affected by ARMS knockdown. The results showed that the development of mushroom body and ventral nerve cord are not influenced by ARMS knockdown. However, we found ddaC, one of the class IV da (dendritic arborization) neuron in peripheral nervous system, exhibited decreased dendritic complexity in ARMS knockdown larvae compared with that in control wild-type larvae. Sholl analysis and neurolucida analysis further highlights ARMS RNAi-mediated decreased dendritic complexity results from decreased dendritic end numbers caused by less branching in higher branching orders, but not from decreased number of trees emanating from cell body. In addition, the size of cell soma does not change in neurons with RNAi of ARMS. To further understand whether alteration in dendritic complexity is due to dysregulation in the establishment or the maintenance of ddaC dendrites, we observed ddaC at earlier stage, that is, mid-second stage and early-mid third stage. We also demonstrate that the phenotype observed in larvae ddaC is not due to off-target effect of RNAi since that overexpression of ARMS rescued the defect. Given that ARMS is the downstream of ephrin/Eph and is involved in MAPK pathway and probably in PI3K (phosphoinositide 3-kinases) pathway, we examined dendritic complexity of the third instar ddaC in the genetic background of ARMS knockdown combined with altered Eph, or PI3K, or MAPK signaling activity.