Cellular and Molecular Events in Early Leech Development

0314718WeisblatIntellectual merit：这是NSF自1984年以来支持的一个基于成就的更新项目。该研究旨在了解水蛭（环节动物门）在细胞和分子水平上的发育。比较不同动物的发育应该有助于理解现代动物的身体结构是如何从祖先物种进化而来的。近年来，大多数动物门被重组为三个主要的分支：后口虫、外生虫和光虫。大多数无脊椎动物，包括环节动物，属于后者，但我们对发展的大部分理解来自后口动物（脊椎动物，海胆）或外生动物（昆虫，线虫）。对水蛭Helobdella的研究使其成为与其他环节动物（寡毛纲和多毛纲）、其他光栖动物（如扁形虫和软体动物）和“模式”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。本研究的目的是继续研究细胞命运的决定，将反向遗传方法（注射反义morpholino寡核苷酸来敲低基因表达或体外合成的mrna，以异位表达天然基因、荧光蛋白或显性阴性结构）与已有的Helobdella胚胎学技术相结合。具体主题包括：1；Helobdella纳米级基因Hro-nos母系遗传的意义。我们提出验证一种假设，即从母体遗传转录本中翻译的HRO-NOS蛋白水平逐渐下降，控制着个体胚胎干细胞（终成细胞）中胚细胞（节段创始细胞）产生的持续时间。Helobdella早期WNT和NOTCH信号的动态。HRO-WNTA蛋白免疫染色在2细胞期呈动态模式；HRO-WNTA信号似乎在第一次细胞分裂后调节细胞-细胞粘附。Hro-notch转录本的原位染色显示了类似的模式，但时间比HRO-WNTA模式提前了约30分钟。我们建议测试Notch和Wnt信号通路在2细胞阶段相互作用的假设，以研究这种相互作用实现的机制。更广泛的影响：1)PI仍然致力于通过招募和培训妇女和代表性不足群体的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。其中一名在校生是通过“初级转学联盟”（Junior Transfer Consortium）加入实验室的。这个项目旨在识别、招募和指导从社区大学转学到伯克利的理科学生。2002年夏天，PI实验室的一名前本科生和博士后研究员（非裔美国男性）被UCSF医学院录取。2) PI还通过评估各种研究项目的申请人，以及参加各种教师与本科生论坛，努力提高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，他在墨西哥、印度和巴西为研究生及以上的学生组织和教授了关于发育和神经发育的密集互动实验室和讲座课程。

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.