Analysis of alin-42/periodNull Allele Implicates All Three Isoforms in Regulation ofCaenorhabditis elegansMolting and Developmental Timing

Abstract

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.

The Doubletime HomologKIN-20Mainly Regulateslet-7Independently of Its Effects on the Period HomologLIN-42inCaenorhabditis elegans

“…The largest isoform, B, contains sequence found in both the A and C isoforms ( Figure 2D ). Work in other labs has suggested that the LIN-42A and B isoforms are of greater functional importance than LIN-42C ( Tennessen et al 2006 ; Monsalve et al 2011 ; Edelman et al 2016 ). Using an antibody developed against the C terminal region of LIN-42 , we were able to clearly visualize the LIN-42A isoform ( Figure 2E , Figure S4).…”

“…LIN-42 has 3 isoforms ( Figure 2D ) ( Edelman et al 2016 ). The largest isoform, B, contains sequence found in both the A and C isoforms ( Figure 2D ).…”

“…LIN-42 also acts as a transcription factor to regulate expression of a multitude of genes ( Mcculloch and Rougvie 2014 ; Van Wynsberghe and Pasquinelli 2014 ; Van Wynsberghe et al 2014 ; Perales et al 2014 ). In addition, lin-42 is essential for proper developmental timing, molting, and entry into an alternative dauer larval stage ( Tennessen et al 2006 , 2010 ; Monsalve et al 2011 ; Edelman et al 2016 ). Consequently, lin-42 mutant worms show defects in circadian rhythmic activity, and exhibit a dumpy phenotype and precocious alae formation ( Abrahante et al 1998 ; Simonetta et al 2009 ; Van Wynsberghe and Pasquinelli 2014 ).…”

Purine Homeostasis Is Necessary for Developmental Timing, Germline Maintenance and Muscle Integrity in Caenorhabditis elegans

“…In addition to size, adsl-1(∆) also displays a strong delay during post-embryonic development. In C. elegans progression through larval stage and molting cycles is highly regulated, involving oscillations of LIN-42, the orthologue the circadian protein PERIOD (Monsalve et al 2011;Edelman et al 2016;reviewed in Monsalve and Frand, 2012). The developmental changes associated with this progression involve a tight coordination of different cell lineages across the worm's body, and are very well characterized, as are the genetic pathways involved (reviewed in Rougvie and Moss, 2013).…”

Adenylosuccinate Lyase activity in the Purine recycling pathway is essential for developmental timing, germline maintenance and muscle integrity inC. elegans

“…In addition to size, adsl-1(∆) also displays a strong delay during post-embryonic development. In C. elegans progression through larval stage and molting cycles is highly regulated, involving oscillations of LIN-42, the orthologue the circadian protein PERIOD [43,44], reviewed in [45]. The developmental changes associated with this progression involve a tight coordination of different cell lineages across the worm's body, and are very well characterized, as are the genetic pathways involved (reviewed in [46]).…”