Rehm E Jay, Hannibal Roberta L, Chaw R Crystal, Vargas-Vila Mario A, Patel Nipam H
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3140, USA.
Cold Spring Harb Protoc. 2009 Jan;2009(1):pdb.prot5128. doi: 10.1101/pdb.prot5128.
The great diversity of arthropod body plans, together with our detailed understanding of fruit fly development, makes arthropods a premier taxon for examining the evolutionary diversification of developmental patterns and hence the diversity of extant life. Crustaceans, in particular, show a remarkable range of morphologies and provide a useful outgroup to the insects. The amphipod crustacean Parhyale hawaiensis is becoming established as a model organism for developmental studies within the arthropods. This protocol describes the injection of P. hawaiensis blastomeres with fluorescently labeled tracers for the purpose of cell-lineage analysis. The total (holoblastic) cleavages that characterize early embryogenesis in P. hawaiensis generate an eight-cell embryo with a stereotypical arrangement of blastomeres, each of which already possesses an invariant cell fate. Fluorochrome-conjugated dextran solutions, mRNAs encoding fluorescent proteins, and biotin-dextran have all proven to be useful lineage markers. The relative merits of various tracers are considered.
节肢动物身体结构的巨大多样性，以及我们对果蝇发育的详细了解，使节肢动物成为研究发育模式进化多样化以及现存生命多样性的首要类群。特别是甲壳类动物，展现出了显著的形态范围，并为昆虫提供了一个有用的外类群。夏威夷片脚类甲壳动物Parhyale hawaiensis正逐渐成为节肢动物发育研究的模式生物。本方案描述了为进行细胞谱系分析，向夏威夷Parhyale hawaiensis的卵裂球注射荧光标记示踪剂的方法。夏威夷Parhyale hawaiensis早期胚胎发育的特征性全裂产生一个具有卵裂球定型排列的八细胞胚胎，每个卵裂球都已经具有不变的细胞命运。荧光染料偶联的葡聚糖溶液、编码荧光蛋白的mRNA和生物素葡聚糖都已被证明是有用的谱系标记。文中考虑了各种示踪剂的相对优点。
