BMP訊息在澤蛭原腸胚中胚層發育之功能性實驗分析

Abstract

BMP是一群廣為人知且研究深入的訊息傳遞分子，BMP在胚胎發育的過程中扮演了許多重要的角色，而胚胎背腹體軸的決定是其中最有名的一種。先前研究發現胚胎會藉由BMP與其拮抗分子形成的濃度梯度來決定背腹體軸的相對位置，許多物種橫跨了各種兩側對稱動物都是藉由這種機制來決定背腹體軸，顯示出這樣的體軸決定方式在演化上是十分保守的。在澤蛭的胚胎原腸化時期，外胚層同樣是利用BMP訊息梯度決定背腹體軸的走向，但所用的BMP並非多數物種所使用的BMP2/4s而是BMP5-8。本實驗主要想要探討這種特別的體軸決定方式是不是也適用在澤蛭的中胚層，我利用不同的方式在中胚層異位表現或抑制BMP的訊息傳遞來觀察對胚胎造成的影響。在異位表現的實驗中我將突變的BMP受器表現在中胚層中，發現此突變會強制開啟下游訊息傳遞，在這些胚胎中，胚胎細胞的細胞遷徙會受到影響，停留在動物極靠近背側的區域，無法完成原腸化。經由後續的實驗發現，此細胞遷徙的缺損是因為異常的微絲細胞骨架所造成。但當過量表現BMP5-8時以及抑制BMP下游訊息傳遞時，細胞的遷徙並未受到影響，因此Hau-BMP5-8並非中胚層細胞在原腸胚中的遷徙所需。接下來我檢驗在對BMP訊息進行操弄後，是否會影響中胚層內背腹體軸的決定。我發現Dachshund和Twist1基因會表現在中胚層的腹側，而在BMP訊息受到異位活化或者抑制的情形下，這些基因的表現位置都沒有受到影響，因此中胚層背腹方向的體軸建立並非受到BMP梯度引導。本研究發現在澤蛭原腸化時期中胚層體軸的決定乃獨立於控制外胚層體軸決定的BMP訊息調控路徑之外。

BMP is a well-known signaling ligand that takes part in many developmental processes during embryogenesis. One of its most important roles in early embryogenesis is in the establishment of body axis. The dorsoventral body axis was found be patterned by the extracellular BMP signaling gradient, arising from the interactions between the BMP ligands and their antagonists. In the leech Helobdella, a non-canonical BMP-mediated dorsoventral patterning mechanism was found to operate in the ectoderm, where BMP5-8 is used as the key BMP ligand that provides the dorsoventral polarity in place of the commonly used BMP2/4 in other species. However, it is not clear whether BMP also patterns the dorsoventral axis of mesoderm. Here, I used various gain-of-function and loss-of-function approaches to determine whether BMP signal participate in the development of mesoderm during the gastrulation stage in the leech. When a constitutively active receptor was mis-expressed in the mesoderm, mesodermal migration was disrupted. However, mis-expression of BMP5-8 produce no comparable migration phenotype. The migration phenotype produced by the mis-expression of constitutively active receptor was caused by abnormal actin dynamics. Knockdown of BMP5-8 or mis-expression of dominant negative receptor produce no obvious cell migration phenotype. To see if BMP signaling is involved in dorsoventral patterning of cell fates in the mesoderm, I examined the expression pattern of Hau-Dach and Hau-Twist1 in embryos in which BMP signaling activity is experimentally manipulated. These marker genes are normally expressed in mesoderm. Disruption of BMP signaling in the mesoderm, however, did not disrupt the expression pattern of genes in the mesoderm, suggesting that BMP signaling is not required for the specification of dorsal and ventral mesoderm fates during gastrulation. Therefore, the migration and patterning of mesoderm in leech gastrula are controlled by mechanism that is independent of the dorsoventral BMP gradient.