研究 bHLH 基因在果蠅眼睛的發育

1. 研究bHLH基因在果蠅眼睛的發育 • 在發育生物學中，一群原本沒有差異的細胞如何能發育成各種具有不同功能的細胞是一門十分重要且有趣的問題。對於研究各個細胞如何藉由訊息傳遞來決定彼此的命運，果蠅的眼睛是一個很好的實驗工具。本篇論文主要探討Atonal (Ato) 與Daughterless (Da) 這兩個basic helix-loop-helix (bHLH) 蛋白質在果蠅眼睛的R8感光細胞的發育上扮演著不可或缺的重要角色。Ato 是一種原神經蛋白質，它會表現於果蠅幼蟲眼睛器官 (eye disc) 中，且其表現量會隨著morphogenetic furrow (MF) 而有所變化。而這些表達 Ato細胞的分佈情形是一個很有趣的現象。在MF之前是所有的細胞都有，但在MF中則是位於規則的細胞群中，而在MF之後則只位於R8的早期細胞。別人的發現 (Gabay, et al.,1997) 指出MAPK在MF的細胞群中被活化，這意味著Ato的表現可能與MAPK的活化有關。跟隨此重要發現，我們進而證明了MAPK的確會抑制Ato的表現，而且MAPK的活化是需要 ato 與da共同來推動產生的。藉著實驗結果我們提立一套ato的負向調控理論來說明為何在MF中只有特定且規則的細胞群會表現ato。為了更進一步的瞭解ato與da在R8感光細胞發育的其他步驟中的功能，我們利用了GAL/UAS實驗技術在不同的區域大量表達ato 與da 。十分有趣地，我們發現大量表達da可以引發R8感光細胞形成，而且此過程不需要ato ，另一方面，MF的移動又需要ato與da的合作。在未來，我們需更進一步的研究。

2. The Function of the bHLH Genes in Drosophila eye development • atonal (ato) encodes a basic-helix-loop-helix (bHLH) protein and is required for the specification of the R8 photoreceptor cells in the Drosophila retinal primordium. The Ato protein is expressed initially in a stripe anterior to the morphogenetic furrow (MF); in the MF this stripe expression is resolved into regularly spaced clusters of Ato-positive cells, the proneural clusters, intervened with Ato-negative cells. Another bHLH protein Daughterless (Da) dimerizes with Ato and is expressed at an enhanced level in Ato expressing cells. Here we show that during the late stage of the proneural clusters, the mitogen activated protein kinase (MAPK) is activated in the proneural clusters. Normal ato or da activity is required for the maintenance of the MAPK activation. Furthermore, in ato or da mutants, the Ato expression is expanded to all cells in the MF, suggesting that ato and da are required for Ato repression in cells between the proneural clusters. By changing the MAPK activity in the proneural clusters, we show that the MAPK activity can mediate the Ato repression nonautonomously. Consistent with this, hyperactivation of MAPK in a stripe of cells posterior to or overlapping the Ato stripe eliminates the proneural cluster formation. Taken together, these results suggest that a negative regulatory loop involving activation of MAPK in the proneural clusters and downregulation of Ato in cells between the proneural clusters is required for the generation of the regularly spaced proneural clusters.To further understand the function of atonal and daughterless in R8 differentiation and furrow progression, I used GAL/UAS system to express atonal and daughterless in different region of eye disc and I got some interesting phenotype. Firstly, misexpression of da behind the furrow induces ectopic R8 cell differentiation. Secondly, I observed that ectopic expression of ato and da induces ectopic furrow. I demonstrate that ato and da are involved in furrow progesstion, but not initiation. In addition, we have found that ectopic R8 cells may come form other cells (R1-R7), but we need to do further study.