Cellular and Molecular Events in Early Leech Development

WeisblatGlossiphoniid水蚤，如Helobdella robusta，对研究发育过程很有用，原因有两个：首先，它们是相对简单的动物，有大的、可接触的胚胎，适合结合细胞和分子技术。其次，由于水蚤属于环节动物门，这是一个研究较少的类群，所获得的结果对进行系间比较是有用的。这样的比较对于区分一般的发育过程和特定的门的发育过程是至关重要的，也是为了了解发育过程的进化修饰如何导致不同动物类型的出现。例如，环节动物和节肢动物彼此相似(而不同于软体动物)，因为它们都有分段的身体平面。但早期Helobdella胚胎与早期果蝇胚胎有显著不同，与软体动物胚胎相似。因此，在早期水蚤胚胎中如何决定细胞命运的问题是相当有意义的。将要进行的实验分为两个主要领域。大多数拟议的努力将用于继续研究细胞命运决定和分化，将反向遗传方法与胚胎学技术相结合，Helobdella胚胎非常适合这种技术。具体研究内容包括：1.中胚层-外胚层命运的决定。魏斯布拉特博士和他的研究小组将测试一种假设，即母系遗传的HRO-nos转录产物--Helobdella中的Nanos同源物--只在胚胎的动物极部被翻译，以及遗传高水平的HRO-NOS蛋白会导致大麦D‘的动物女儿承担外胚层命运。2.生殖系的起源。HRO-nos也是合子转录的。与其他双边动物一样，水蚤的HRO-nos与原始生殖细胞有关的假设也将得到验证。3.肠道形成过程中的刺猬类基因产物HRO-HH的信号传导。初步结果表明，来自内胚层的HRO-HH信号在水蚤和脊椎动物的肠道形成中起着关键作用。将进行实验以证实这一结果，并寻找HRO-NOS信号的下游靶点。第二部分工作需要研究细胞动力学和细胞过程。具体的研究课题包括：1.细胞-细胞融合的调控。水蚤中肠形成的早期步骤需要大单体A“”和B“”的融合，这一过程由胚胎D象限发出的信号调节。将进行卵裂球分离和重组实验，以确定信号的时间和来源。2.早期WNT信号的动态变化。Weisblat博士的团队最近发现，WNT信号通路在水蚤的2-细胞胚胎中起作用，并在一定程度上调节第一次细胞分裂后的细胞-细胞黏附。这是细胞间胚胎信号传递的最早的例子。所提出的实验旨在确定WNT的表达是否在转录、翻译或翻译后处理水平上受到调控。

0091261WeisblatGlossiphoniid leeches such as Helobdella robusta, are useful for studying developmental processes for two reasons: first, they are relatively simple animals with large, accessible embryos that are suited for combined cellular and molecular techniques. Second, since leeches belong to the phylum Annelida, an otherwise poorly studied group, the results obtained are useful for making interphyletic comparisons. Such comparisons are essential to distinguish general developmental processes from phylum-specific ones, and also to learn how evolutionary modification of developmental processes leads to the appearance of diverse animal types. For example, annelids and arthropods resemble each other (and differ from mollusks) in that they both have segmented body plans. But the early Helobdella embryo is strikingly different from the early Drosophila embryo and similar to molluscan embryos. Thus, the question of how cell fates are determined in the early leech embryo is of considerable interest. The experiments to be undertaken fall into two main areas. Most of the proposed effort will go into continuing investigations of cell fate determination and differentiation, combining reverse genetic approaches with embryological techniques for which the Helobdella embryo is well suited. Specific topics to be investigated include: 1. The mesoderm-ectoderm fate decision. Dr. Weisblat and his research group will test the hypothesis that maternally inherited transcripts of Hro-nos, the nanos homolog in Helobdella, are translated only at the animal pole of the embryo, and that inheriting high levels of HRO-NOS protein causes the animal daughter of macromere D' to assume an ectodermal fate. 2. The origins of the germline. Hro-nos is also transcribed zygotically. The hypothesis that Hro-nos is associated with primordial germ cells in leech, as in other bilaterians will also be tested. 3. Signalling by HRO-HH, a hedgehog-class gene product in gut formation. Preliminary results indicate that HRO-HH signaling from endoderm is critical for gut formation in leech as in vertebrate. Experiments will be undertaken to confirm this result and look for downstream targets of HRO-NOS signaling.The second part of the work entails investigations of the dynamics of cells and cellular processes. Specific topics to be investigated include: 1. The regulation of cell-cell fusion. An early step in leech midgut formation entails the fusion of macromeres A''' and B''', a process which is regulated by signals emanating from the D quadrant of the embryo. Blastomere isolation and recombination experiments will be performed to pinpoint the timing and source of the signal. 2. The dynamics of early WNT signalling. Dr. Weisblat's group has recently discovered that a WNT signalling pathway is operative in the 2-cell embryo of the leech, and serves in part to regulate cell-cell adhesion following the first cell division. This is the earliest possible example of intercellular embryonic signalling. The experiments proposed are designed to determine whether WNT expression is regulated at the level of transcription, translation, or post-translational processing.