Cellular and Molecular Events in Early Leech Development

0314718 Weisblat智力价值：这是一个基于成就的项目，自1984年以来一直由NSF支持。这项研究旨在了解水蛭（环节动物门）在细胞和分子水平上的发育。比较各种动物的发育过程有助于理解现代动物的体型是如何从祖先物种进化而来的。近年来，大多数动物门重新划分为三个主要分支，即后口门、蜕皮动物门和散轮动物门。 大多数无脊椎动物门，包括环节动物门，属于后一组，但我们对发育的大多数理解来自半口动物门（脊椎动物，海胆）或蜕皮动物门（昆虫，线虫）。对水蛭Helobdella的研究使其成为与其他环节动物（寡毛类和多毛类），其他lophotrochozoans（例如扁形虫和软体动物）和“模型”系统进行比较的有用出发点。 Helobdella对研究发育很有用，因为它的胚胎适合细胞和分子技术。这项建议是继续调查的细胞命运的决定，结合反向遗传方法（注射反义吗啉代寡核苷酸敲低基因表达或在体外合成的mRNA异位表达天然基因，荧光蛋白或显性负结构）与胚胎学技术已经可用于Helobdella。 具体议题包括：1. Helobdella nanos类基因Hro-nos的母系遗传意义 我们建议测试这一假设，即从母系遗传的转录本翻译的HRO-NOS蛋白水平逐渐下降，控制个体胚胎干细胞（终末细胞）中胚细胞（节段性创始细胞）产生的持续时间。Helobdella早期WNT和NOTCH信号的动态。 HRO-WNTA蛋白的免疫染色揭示了在2-细胞阶段的动态模式; HRO-WNTA信号转导似乎在第一次细胞分裂后调节细胞-细胞粘附。 Hro-缺口转录物的原位染色显示出类似的模式，但时间比HRO-WNTA模式提前约30分钟。 本研究旨在验证Notch和Wnt信号通路在2-细胞阶段相互作用的假设，并探讨其相互作用的机制。更广泛的影响：1）PI仍然致力于通过招募和培训女性和代表性不足的群体成员来扩大国家科学人才库。目前，实验室小组由1名助理研究员（女性）、5名研究生（3名女性，包括1名墨西哥/美洲原住民）和2名本科生（均为菲律宾裔）组成。 目前的本科生之一，通过“少年转学联盟”加入了实验室，该联盟是一个旨在识别、招募和指导从社区学院以初级身份转学到伯克利的理科学生的计划。PI实验室的一名前本科生和博士后研究员（非裔美国男性）于2002年夏天被UCSF医学院录取。 2)PI还致力于通过评估各种研究项目的申请人以及参加各种学院本科生论坛来增强这所大型研究型大学的本科生体验。 3)PI积极促进美国和发展中国家之间的科学善意和合作。 为此，他帮助组织和教授密集和互动的实验室和讲座课程的发展和神经发育，在研究生水平和超越墨西哥，印度和巴西的学生。

0314718WeisblatIntellectual merit: This is an Accomplishment-Based Renewal of a project supported by NSF since 1984. The research seeks to understand development at the cellular and molecular levels in leeches (phylum Annelida). Comparing development in various animals should contribute to understanding how body plans of modern animals evolved from those of ancestral species.Recent years have seen the reorganization of most animal phyla into three main clades, Deuterostomia, Ecdysozoa and Lophotrochozoa. Most invertebrate phyla, including Annelida, belong the latter group, but most of our understanding of development has come from Deuterostomia [vertebrates, sea urchins] or Ecdysozoa [insects, nematodes]. Work on the leech Helobdella make it a useful point of departure for comparisons with other annelids (oligochaetes and polychaetes), other lophotrochozoans (e.g. flatworms and mollusks) and the "model" systems. Helobdella is useful for studying development because its embryos are suited for cellular and molecular techniques. This proposal is to continue investigations of cell fate determination, combining reverse genetic approaches (injecting anti-sense morpholino oligonucleotides to knock down gene expression or mRNAs synthesized in vitro to ectopically express native genes, fluorescent proteins or dominant negative constructs) with embryological techniques already available for Helobdella. Specific topics include:1. Significance of the maternally inherited Hro-nos, a nanos-class gene in Helobdella . We propose to test the hypothesis that gradually declining levels of HRO-NOS protein, translated from maternally inherited transcripts, control the duration of blast cell (segmental founder cell) production in individual embryonic stem cells (teloblasts).2. The dynamics of early WNT and NOTCH signaling in Helobdella. Immunostaining for HRO-WNTA protein reveals a dynamic pattern during the 2-cell stage; HRO-WNTA signaling seems to regulate cell-cell adhesion following the first cell division. In situ staining for Hro-notch transcripts shows a similar pattern, but time shifted about 30 minutes ahead of the HRO-WNTA pattern. We propose to test the hypothesis that Notch and Wnt signaling pathways interact in the 2-cell stage, to investigate the mechanisms by which this interaction is achieved.Broader impacts: 1) The PI remains committed to expanding the national pool of scientific talent by recruiting and training women and members of underrepresented groups. Currently, the lab group consists of 1 assistant researcher (woman), 5 graduate students (3 women, including 1 of Mexican/native American origins) and 2 undergraduates (both of Filipino origin). One of the current undergrads, joined the lab via the "Junior Transfer Consortium", a program to identify, recruit and mentor science students who transfer to Berkeley with junior status from community colleges. A previous undergrad and postbac researcher (African-American male) from the PI's lab matriculated at UCSF medical school in the summer of 2002. 2) The PI also works to enhance the undergraduate experience at this large, research university by evaluating applicants for various research programs, and by participating in various faculty-undergrad forums. 3) The PI is active in promoting scientific goodwill and collaborations between the USA and developing nations. To this end, he has helped to organize and teach intensive and interactive lab and lecture courses in development and neurodevelopment, for students at the graduate level and beyond in Mexico, India and Brazil.