Cellular and Molecular Events in Early Leech Development

0314718 WeisblatIntelligence功绩：这是自1984年以来由NSF资助的一个基于成就的项目的续订。这项研究试图在细胞和分子水平上了解水蚤(环节动物门)的发育。比较不同动物的发育有助于理解现代动物的身体平面是如何从祖先物种的身体平面进化而来的。近年来，大多数动物门被重组为三个主要支系，后口纲、蜕皮纲和线虫。大多数无脊椎动物门，包括环节动物，属于后一类，但我们对发育的大部分了解来自后口纲[脊椎动物，海胆]或蜕皮虫[昆虫，线虫]。对水蚤Helobdella的研究使其成为与其他环节动物(寡毛类和多毛类)、其他轮虫(例如扁虫和软体动物)和“模式”系统进行比较的有用起点。Helobdella对研究发育很有用，因为它的胚胎适合细胞和分子技术。这一建议是继续研究决定细胞命运的方法，结合已有的胚胎学技术，结合反向遗传方法(注射反义吗啉寡核苷酸以下调基因表达或在体外合成mRNAs以异位表达天然基因、荧光蛋白或显性负结构)。具体的研究主题包括：1.Helobdella中的Nanos类基因--母系遗传的HRO-nos的意义。我们建议检验这一假设，即从母系遗传的转录本翻译而来的HRO-NOS蛋白水平逐渐下降，控制单个胚胎干细胞(成体细胞)中原始细胞(节段性创始人细胞)产生的持续时间。Helobdella早期WNT和NOTCH信号的动态。HRO-WNTA蛋白的免疫染色显示了2-细胞阶段的动态模式；HRO-WNTA信号似乎在第一次细胞分裂后调节细胞与细胞的黏附。HRO-Notch转录本的原位染色显示出类似的模式，但时间比HRO-WNTA模式提前了约30分钟。我们建议检验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。广泛的影响：1)PI仍然致力于通过招聘和培训女性和代表性不足群体的成员来扩大国家的科学人才库。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名墨西哥裔/美洲原住民)和2名本科生(均为菲律宾裔)组成。作为目前的本科生之一，他通过“初级转学联盟”加入了实验室，这是一个旨在识别、招募和指导从社区大学转学到伯克利的初级理科学生的计划。PI实验室以前的本科生和后BAC研究员(非裔美国男性)于2002年夏天进入加州大学旧金山分校医学院学习。2)PI还致力于通过评估各种研究项目的申请者，以及参与各种教职员工与本科生的论坛，来加强这所大型研究型大学的本科生体验。3)国际和平研究所积极促进美国和发展中国家之间的科学善意和合作。为此，他帮助墨西哥、印度和巴西的研究生和研究生组织和教授发展和神经发育方面的密集和互动的实验室和讲座课程。

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.