Edelman Theresa L B, McCulloch Katherine A, Barr Angela, Frøkjær-Jensen Christian, Jorgensen Erik M, Rougvie Ann E
Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota 55454.
Department of Biology, Howard Hughes Medical Institute, University of Utah, Salt Lake City, Utah 84112.
G3 (Bethesda). 2016 Dec 7;6(12):4077-4086. doi: 10.1534/g3.116.034165.
The Caenorhabditis elegans heterochronic gene pathway regulates the relative timing of events during postembryonic development. lin-42, the worm homolog of the circadian clock gene, period, is a critical element of this pathway. lin-42 function has been defined by a set of hypomorphic alleles that cause precocious phenotypes, in which later developmental events, such as the terminal differentiation of hypodermal cells, occur too early. A subset of alleles also reveals a significant role for lin-42 in molting; larval stages are lengthened and ecdysis often fails in these mutant animals. lin-42 is a complex locus, encoding overlapping and nonoverlapping isoforms. Although existing alleles that affect subsets of isoforms have illuminated important and distinct roles for this gene in developmental timing, molting, and the decision to enter the alternative dauer state, it is essential to have a null allele to understand all of the roles of lin-42 and its individual isoforms. To remedy this problem and discover the null phenotype, we engineered an allele that deletes the entire lin-42 protein-coding region. lin-42 null mutants are homozygously viable, but have more severe phenotypes than observed in previously characterized hypomorphic alleles. We also provide additional evidence for this conclusion by using the null allele as a base for reintroducing different isoforms, showing that each isoform can provide heterochronic and molting pathway activities. Transcript levels of the nonoverlapping isoforms appear to be under coordinate temporal regulation, despite being driven by independent promoters. The lin-42 null allele will continue to be an important tool for dissecting the functions of lin-42 in molting and developmental timing.
秀丽隐杆线虫异时性基因通路调控胚胎后发育过程中事件的相对时间。lin-42是生物钟基因period的线虫同源物，是该通路的关键元件。lin-42的功能已由一组导致早熟表型的亚效等位基因所定义，在这些表型中，诸如皮下细胞的终末分化等后期发育事件发生得过早。一部分等位基因还揭示了lin-42在蜕皮过程中的重要作用；在这些突变动物中，幼虫阶段延长且蜕皮常常失败。lin-42是一个复杂的基因座，编码重叠和非重叠的异构体。尽管现有的影响异构体亚组的等位基因已经阐明了该基因在发育时间、蜕皮以及进入替代滞育状态的决定中的重要且独特的作用，但要了解lin-42及其各个异构体的所有作用，拥有一个无效等位基因至关重要。为了解决这个问题并发现无效表型，我们构建了一个删除整个lin-42蛋白质编码区域的等位基因。lin-42无效突变体是纯合可存活的，但具有比先前表征的亚效等位基因中观察到的更严重的表型。我们还通过将无效等位基因作为重新引入不同异构体的基础提供了这一结论的额外证据，表明每个异构体都可以提供异时性和蜕皮通路活性。尽管由独立启动子驱动，但非重叠异构体的转录水平似乎受到协调的时间调控。lin-42无效等位基因将继续是剖析lin-42在蜕皮和发育时间中的功能的重要工具。
