Materials and Methods Figs. S1 to S4 Tables S1 to S5 References and Notes Micrornas Regulate Brain Morphogenesis in Zebrafish-猫眼课题宝

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A Schlesinger;A. Kiger;N. Perrimon;B. Z. Shilo;Dev;D. Sinner;S. Rankin;M. Lee;A. M. Zorn

对手稿进行热烈的讨论和评论。我们感谢D. J. Grau，用于Rab 5表达构建体的辅助。我们也感谢果蝇RNAi筛选中心（DRSC）和化学与细胞生物学研究所（ICCB）的成员的帮助。感谢G。Weidinger协助斑马鱼实验和J. Tee协助构建siRNA和cDNA表达构建体。R.D. microRNAs（miRNAs）是一类转录后调节基因表达的小分子RNA。为了阻断斑马鱼中所有miRNA的形成，我们产生了破坏Dicer核糖核酸酶III和双链RNA结合结构域的母合子Dicer（MZdicer）突变体。突变体胚胎不将前体miRNA加工成成熟的miRNA，但是注射预加工的miRNA恢复基因沉默，这表明被破坏的结构域在沉默的后续步骤中被保留。MZdicer突变体经历轴形成和分化多种细胞类型，但在原肠胚形成、脑形成、体节发生和心脏发育期间显示异常形态发生。注射miR-430 miRNA挽救了MZdicer突变体中的脑缺陷，揭示了miRNA在形态发生过程中的重要作用。microRNA是进化上保守的小的非蛋白质编码RNA基因产物，在转录后水平调节基因表达（1-3）。在动物中，成熟的miRNA是1022个核苷酸（nt）长，并且通过属于核糖核酸酶III（RNaseIII）家族的核酸酶的连续加工从初级转录物（称为pri-miRNA）产生。首先，Drosha切割pri-miRNA并切除约70 nt的茎环前体（称为pre-miRNA），然后用Dicer切割（4-7）。经加工的双链体的一条链被掺入沉默复合物中，并将其引导至靶序列（1，3）。这导致靶mRNA的切割和/或其生产性翻译的抑制（1-3）。已经预测或鉴定了数百种脊椎动物miRNA和数千种miRNA靶点，但对发育过程中miRNA的功能知之甚少（1，2，8，9）。脊椎动物miRNA功能的线索来自几种方法，这些研究表明脊椎动物miRNA可能参与干细胞维持（12，19）或细胞命运决定等过程（17，18，20）;然而，没有功能丧失分析指定特定miRNA或miRNA家族在体内的作用，目前还不清楚miRNAs在脊椎动物胚胎发生过程中的作用有多广泛。揭示脊椎动物miRNA的全局作用的一种方法是使用切丁酶突变体来消除成熟miRNA的产生。例如，切丁...

for lively discussion and critical comments on the manuscript. We thank D. J. Grau for assistance with Rab5 expression constructs. We also thank members of the Drosophila RNAi Screening Center (DRSC) and Institute of Chemistry and Cell Biology (ICCB) for their assistance. We thank G. Weidinger for assistance with zebrafish experiments and J. Tee for assistance with construction of siRNA and cDNA expression constructs. R.D. MicroRNAs (miRNAs) are small RNAs that regulate gene expression posttranscriptionally. To block all miRNA formation in zebrafish, we generated maternal-zygotic dicer (MZdicer) mutants that disrupt the Dicer ribonuclease III and double-stranded RNA–binding domains. Mutant embryos do not process precursor miRNAs into mature miRNAs, but injection of preprocessed miRNAs restores gene silencing, indicating that the disrupted domains are dispensable for later steps in silencing. MZdicer mutants undergo axis formation and differentiate multiple cell types but display abnormal morphogenesis during gastrulation, brain formation, somito-genesis, and heart development. Injection of miR-430 miRNAs rescues the brain defects in MZdicer mutants, revealing essential roles for miRNAs during morphogenesis. MicroRNAs are evolutionarily conserved small non–protein-coding RNA gene products that regulate gene expression at the posttran-scriptional level (1–3). In animals, mature miRNAs are È22 nucleotides (nt) long and are generated from a primary transcript (termed pri-miRNA) through sequential processing by nucleases belonging to the ribo-nuclease III (RNaseIII) family. Initially, Drosha cleaves the pri-miRNA and excises a stem-loop precursor of È70 nt (termed pre-miRNA), which is then cleaved by Dicer (4–7). One strand of the processed duplex is incorporated into a silencing complex and guides it to target sequences (1, 3). This results in the cleavage of target mRNAs and/or the inhibition of their productive translation (1–3). Several hundred vertebrate miRNAs and several thousand miRNA targets have been predicted or identified, but little is known about miRNA function during development (1, 2, 8, 9). Clues to vertebrate miRNA function have come from several approaches, These studies have led to the suggestions that vertebrate miRNAs might be involved in processes such as stem cell maintenance (12, 19) or cell fate determination (17, 18, 20); however , no loss-of-function analysis has assigned a role for a particular miRNA or miRNA family in vivo, and it has been unclear how widespread the role of miRNAs is during vertebrate embryogenesis. One approach to reveal the global role of vertebrate miRNAs is to abolish the generation of mature miRNAs with the use of dicer mutants. For example, dicer …