Netrin訊息分子在澤蛭胚胎原腸化之功能

Abstract

Netrin是一個被分泌至細胞外的蛋白質，過去已知Netrin的功能包括軸突的導引以及細胞的遷徙。在澤蛭中有兩個netrin基因：Hau-netrin1和Hau-netrin2，這兩個netrin基因都在germinal band形成以及原腸化的時期就有表現，澤蛭胚胎的germinal band主要由五個不同teloblast進行不對稱分裂後產生的子細胞排列形成。一開始，來自不同teloblast的細胞會在以動物極為中心的micromere cap後緣開始進行組裝，接著由後端teloblast新產生的細胞會將前端的細胞向前端的腹側推送，使左右兩邊的germinal bands在腹面的中線由前往後癒合形成germinal plate。本實驗主要探討表現在神經外胚層(N)上的Hau-netrin2在germinal band形成所扮演的角色，首先我試圖以剔除表現Hau-netrin2的細胞或是antisense morpholino來knock-down Hau-netrin2的表現觀察實驗對胚胎型態生成的影響。雖然morpholino knock-down的方式並沒有辦法造成胚胎在形態上顯著的差異，但是如果將N teloblast 剔除卻會造成中胚層的細胞移動到另外一側的germinal band上。另一方面，在進行基因過量表現的實驗中，將Hau-netrin2異位表現在中胚層以及其他外胚層上皆會造成中胚層細胞不正常的遷徙。而從中胚層細胞移動的形態以及負責吸引功能的netrin受體的表現形態可以推論Hau-netrin2 是一個吸引中胚層細胞的訊息分子。本實驗發現在germinal band生成時，外胚層的細胞能夠利用netrin訊息分子引導中胚層細胞的移動。

Netrin is a secreted signaling molecule which is known to be involved in axon guidance and cell migration. Here, in the leech Helobdella, two netrin genes, Hau-netrin1 and Hau-netrin2, were identified, and the leech netrins were expressed in early development during germinal band assembly and gastrulation. Germinal bands consist of five columns of blast cells produced by the asymmetric divisions of teloblasts. In the beginning, blast cells from different lineages are assembled into a germinal band at the posterior edge of the micromere cap which occupies the animal pole territory, and then the posteriorly added blast cells push the anterior cells toward the prospective anterior ventral midline, where the left and right germinal bands coalesce progressively to form germinal plates. This study focuses on the roles of Hau-netrin2 which is continuously expressed in the N lineages contributing to the ventral neuroectoderm during germinal band formation and gastrulation. First, I attempted the cell ablation experiment and the loss-of-function study by morpholino-mediated knock-down. Although injection of antisense morpholino did not produce any significant phenotype, the mesodermal (M) lineages were switched to the contralateral germinal band after the N lineages which produced Hau-netrin2 were eliminated. In the gain-of-function studies, ectopic expression of Hau-netrin2 mRNA in the mesoderm or dorsal ectoderm also perturbed the migration of the M lineages. Based on the migration patterns of the M lineages and the expression pattern of the netrin receptor, my results suggest that Hau-netrin2 produced by N lineages is an attractive cue for the migration of M lineages. This study uncovers a role for netrin signaling in coordinating the movement of different germ layers in leech gastrulation.