Analysis of a lin-42/period Null Allele Implicates All Three Isoforms in Regulation of Caenorhabditis elegans Molting 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.

Keywords: Caenorhabditis elegans; heterochrony; lin-42; molting.

Figure 1

lin-42(ox461) deletes the lin-42 coding region. (A) lin-42 genomic locus and transcription units. The line with terminal arrowheads represents genomic DNA of the lin-42 locus drawn with 5′ to the left, which is inverted from WormBase. lin-42 alleles and the Mos1 insertion (ttTi42556) are indicated above the line. SacI and EcoRI sites are included for reference with (B) and (C), but not all recognition sites for these enzymes are shown. The lin-42 locus produces three transcription units diagrammed below, with filled boxes representing exons: lin-42a, lin-42b, and lin-42c. lin-42a and lin-42c are nonoverlapping and expressed from distinct promoters (Tennessen et al. 2006). Note that the lin-42 nomenclature used here conforms to that adopted by WormBase and differs from that of pre-2014 publications from the Rougvie laboratory (e.g., Tennessen et al. 2006, 2010). (B) lin-42 deletion alleles. The extent of each deletion is noted in brackets. The lin-42(n1089) PASA domain is in parentheses since the majority of the domain is deleted. (C) The lin-42(ox461) allele deletes the lin-42 coding region and replaces it with Punc-122:gfp and C. briggsae unc-119(+). The fragments used as repair templates in creation of the deletion allele are indicated. See Materials and Methods for details.

Figure 2

lin-42(0) animals exhibit highly penetrant larval arrest and developmental delay phenotypes. (A) lin-42(0) mutants have a severe larval arrest phenotype. Individually plated wild-type, lin-42(n1089), lin-42(ok2385), and lin-42(ox461) mutants were monitored for developmental progression. The percentage of animals that arrested as larvae and failed to attain adulthood by 8 d posthatching is shown. n ≥ 100 for each genotype. (B) Micrographs of wild-type and lin-42(ox461) animals 72 hr posthatching. At this time point, all wild-type animals had reached adulthood and were laying eggs, whereas <2% of lin-42(0) mutants were adults, and they did not yet contain fertilized eggs, only oocytes. Most lin-42(0) animals appeared unable to complete the second larval molt. The left lin-42(0) panel contains two animals that are the same chronological age, with an inset showing the smaller animal is trapped in an unshed cuticle (white arrow). The right panel shows a 96 hr animal that appears arrested by size, but has nevertheless begun vulval morphogenesis, marked by the white v in inset. (C) Timing of molts in seven representative wild-type animals (top) and lin-42(0) mutants (bottom). Each horizontal row represents an individual animal that was monitored for pharyngeal pumping, lethargus, and ecdysis. Gray shading denotes animals in lethargus and vertical red lines indicate that a shed cuticle was observed on the plate. A total of 68 animals were followed for lin-42(ox461) and 10 for wild type. (D) The time of adult onset is delayed in lin-42 mutants. Animals from (A) that bypassed larval arrest were scored for the day posthatching that they reached adulthood, and graphed as percentage of animals that escaped arrest. n = 20, 125, 60, and 23 for wild type, n1089, ok2385, and ox461, respectively.

Figure 3

lin-42(0) mutants have a precocious heterochronic phenotype. (A) lin-42 mutations cause precocious seam cell development. Shown is a representative seam cell lineage (V1–V4, V6) diagram in wild-type and lin-42(0) mutants. A horizontal line indicates a cell division and triple horizontal bars indicate alae formation. Developmental stages are relative to the molts, and the actual intermolt periods of wild-type and lin-42(0) animals are different (see Figure 2C). (B) lin-42(0) mutants have a more severe heterochronic defect than lin-42(lf) animals. Wild-type, lin-42(n1089), lin-42(ok2385), and lin-42(ox461) animals were analyzed at the L3 molt for alae formation. Animals were scored for either complete, partial (alae with gaps), or no alae. n ≥ 20 for each strain. **P < 0.0001, Fisher’s exact test. (C) lin-42(0) animals form precocious alae at the L3 molt stage. Micrographs of representative wild-type and lin-42(0) animals at the third molt are shown, with an image of cuticle in the top row and vulval morphogenesis (v) of the animal below to denote staging. Bar, 10 µm applies to all panels.

Figure 4

Levels of three lin-42 transcripts cycle in unison. (A) lin-42 transcript diagrams with small boxes above indicating the locations of primer sets used in qPCR assays: lin-42a (red), lin-42b (green), and lin-42c (purple). lin-42a and lin-42b assays are intron spanning and recognize a single transcript, whereas lin-42c does not contain a unique intron; its primer could also amplify the lin-42b primary transcript. (B–D) Representative time courses of lin-42a, lin-42b, and lin-42c accumulation, relative to the time of mlt-10 expression, in wild-type and lin-42 mutants. Two independent biological replicas for each genotype are shown in Figure S1, along with primer controls. (B) Wild type with time points normalized to 0 hr. (C and D) lin-42a and lin-42c levels cycle in lin-42 mutant backgrounds. Time points within each assay are normalized to 6 hr. (C) lin-42a message levels in lin-42(n1089) mutants. (D) lin-42c message levels in lin-42(ok2385) mutants from 6 to 24 hr.