Developmental function and state transitions of a gene expression oscillator in Caenorhabditis elegans
2020; Springer Nature; Volume: 16; Issue: 7 Linguagem: Inglês
10.15252/msb.20209498
ISSN1744-4292
AutoresMilou W.M. Meeuse, Yannick P. Hauser, Lucas J Morales Moya, Gert‐Jan Hendriks, Jan Eglinger, Guy Bogaarts, Charisios D. Tsiairis, Helge Großhans,
Tópico(s)Circadian rhythm and melatonin
ResumoArticle20 July 2020Open Access Developmental function and state transitions of a gene expression oscillator in Caenorhabditis elegans Milou WM Meeuse orcid.org/0000-0001-6382-6449 Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland University of Basel, Basel, Switzerland Search for more papers by this author Yannick P Hauser orcid.org/0000-0003-1357-9712 Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland University of Basel, Basel, Switzerland Search for more papers by this author Lucas J Morales Moya Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland Search for more papers by this author Gert-Jan Hendriks orcid.org/0000-0001-8798-1443 Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland University of Basel, Basel, Switzerland Search for more papers by this author Jan Eglinger orcid.org/0000-0001-7234-1435 Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland Search for more papers by this author Guy Bogaarts orcid.org/0000-0002-3456-239X University Hospital, Basel, Switzerland Search for more papers by this author Charisios Tsiairis orcid.org/0000-0002-9788-9875 Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland Search for more papers by this author Helge Großhans Corresponding Author [email protected] orcid.org/0000-0002-8169-6905 Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland University of Basel, Basel, Switzerland Search for more papers by this author Milou WM Meeuse orcid.org/0000-0001-6382-6449 Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland University of Basel, Basel, Switzerland Search for more papers by this author Yannick P Hauser orcid.org/0000-0003-1357-9712 Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland University of Basel, Basel, Switzerland Search for more papers by this author Lucas J Morales Moya Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland Search for more papers by this author Gert-Jan Hendriks orcid.org/0000-0001-8798-1443 Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland University of Basel, Basel, Switzerland Search for more papers by this author Jan Eglinger orcid.org/0000-0001-7234-1435 Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland Search for more papers by this author Guy Bogaarts orcid.org/0000-0002-3456-239X University Hospital, Basel, Switzerland Search for more papers by this author Charisios Tsiairis orcid.org/0000-0002-9788-9875 Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland Search for more papers by this author Helge Großhans Corresponding Author [email protected] orcid.org/0000-0002-8169-6905 Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland University of Basel, Basel, Switzerland Search for more papers by this author Author Information Milou WM Meeuse1,2,‡, Yannick P Hauser1,2,‡, Lucas J Morales Moya1, Gert-Jan Hendriks1,2, Jan Eglinger1, Guy Bogaarts3, Charisios Tsiairis1 and Helge Großhans *,1,2 1Friedrich Miescher Institute for Biomedical Research (FMI), Basel, Switzerland 2University of Basel, Basel, Switzerland 3University Hospital, Basel, Switzerland ‡These authors contributed equally to this work *Corresponding author. Tel: +41 61 697 6580; E-mail: [email protected] Mol Syst Biol (2020)16:e9498https://doi.org/10.15252/msb.20209498 Correction(s) for this article Developmental function and state transitions of a gene expression oscillator in Caenorhabditis elegans26 October 2020 Published research reagents from the FMI are shared with the academic community under a Material Transfer Agreement (MTA) having terms and conditions corresponding to those of the UBMTA (Uniform Biological Material Transfer Agreement). PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Gene expression oscillators can structure biological events temporally and spatially. Different biological functions benefit from distinct oscillator properties. Thus, finite developmental processes rely on oscillators that start and stop at specific times, a poorly understood behavior. Here, we have characterized a massive gene expression oscillator comprising > 3,700 genes in Caenorhabditis elegans larvae. We report that oscillations initiate in embryos, arrest transiently after hatching and in response to perturbation, and cease in adults. Experimental observation of the transitions between oscillatory and non-oscillatory states at high temporal resolution reveals an oscillator operating near a Saddle Node on Invariant Cycle (SNIC) bifurcation. These findings constrain the architecture and mathematical models that can represent this oscillator. They also reveal that oscillator arrests occur reproducibly in a specific phase. Since we find oscillations to be coupled to developmental processes, including molting, this characteristic of SNIC bifurcations endows the oscillator with the potential to halt larval development at defined intervals, and thereby execute a developmental checkpoint function. Synopsis The authors investigate a putative developmental clock in C. elegans. Population- and single animal-based analyses uncover a gene expression oscillator that may support a developmental checkpoint function. Extensive rhythmic gene expression in C. elegans larvae is initiated in embryos and is coupled to molting. The oscillator is arrested in a specific phase (normally observed at molt exit) in adults, early L1 and dauer larvae. A bifurcation of the oscillator constitutes a putative developmental checkpoint mechanism. Characteristics of oscillation onset and offset constrain potential oscillator mechanisms as well as mathematical models and their parameters. Introduction Gene expression oscillations occur in many biological systems as exemplified by circadian rhythms in metabolism and behavior (Panda et al, 2002), vertebrate somitogenesis (Oates et al, 2012), plant lateral root branching (Moreno-Risueno et al, 2010), and Caenorhabditis elegans larval development (Hendriks et al, 2014). They are well-suited for timekeeping, acting as molecular clocks that can provide a temporal, and thereby also spatial, structure for biological events (Uriu, 2016). This structure may represent external time, as illustrated by circadian clocks, or provide temporal organization of internal processes without direct reference to external time, as illustrated by somitogenesis clocks (Rensing et al, 2001). Depending on these distinct functions, oscillators require different properties. Thus, robust representation of external time requires a stable period; i.e., the oscillator has to be compensated for variations in temperature and other environmental factors. It also benefits from a phase-resetting mechanism to permit realignments, if needed, to external time. Intuitively, either feature seems unlikely to benefit developmental oscillators. By contrast, because developmental processes are finite; e.g., an organism has a characteristic number of somites, developmental oscillators need a start and an end. How such changes in oscillator activity occur in vivo, and which oscillator features enable them, is largely unknown (Riedel-Kruse et al, 2007; Shih et al, 2015). Here, we characterize the recently discovered "C. elegans oscillator" (Kim et al, 2013; Hendriks et al, 2014) at high temporal resolution and across the entire period of C. elegans development, from embryo to adult. The system is marked by a massive scale where ~3,700 genes exhibit transcript level oscillations that are detectable, with large, stable amplitudes and widely dispersed expression peak times (i.e., peak phases), in lysates of whole animals. For the purpose of this study, and because insufficient information exists on the identities of core oscillator versus output genes, we define the entire system of oscillating genes as "the oscillator". We demonstrate that the oscillations are coupled to molting, i.e., the cyclical process of new cuticle synthesis and old cuticle shedding that occurs at the end of each larval stage. We observe and characterize onset and offset of oscillations both during continuous development and upon perturbation, and find that transitions occur with a sudden change in amplitude. They also occur in a characteristic oscillator phase and thus at specific, recurring intervals. The transitions are a manifestation of a bifurcation, i.e., qualitative change in behavior, of the underlying oscillator system. Hence, our observations constrain possible oscillator architectures, excluding a simple negative-loop design, and parametrization of mathematical models. Functionally, because of the phase-locking of the oscillator and molting, arrests always occur at the same time during larval stages, around molt exit. This time coincides with the previously reported recurring window of activity of a checkpoint that can halt larval development in response to nutritionally poor conditions. Hence, our results indicate that the C. elegans oscillator functions as a developmental clock whose architecture supports a developmental checkpoint function. Results Thousands of genes with oscillatory expression during the four larval stages Although previous reports agreed on the wide-spread occurrence of oscillatory gene expression in C. elegans larvae (Kim et al, 2013; Grün et al, 2014; Hendriks et al, 2014), the published datasets were either insufficiently temporally resolved or too short to characterize oscillations across C. elegans larval development. Hence, to understand the extent and features of these oscillations better, including their continuity throughout development, we performed two extended time course experiments to cover the entire period of post-embryonic development plus early adulthood at hourly resolution. We extracted total RNA from populations of animals synchronized by hatching in the absence of food. The first time course (designated TC1) covered the first 15 h of development on food at 25°C, and the second time course (TC2) covered the span of 5 h through 48 h after plating at 25°C. [Fig EV1A provides a summary of all sequencing time courses analyzed in this study.] The extensive overlap facilitated fusion of these two time courses into one long time course (TC3) (Fig EV1B), and a pairwise-correlation plot of gene expression over time showed periodic similarity (Fig 1A, light-gray off-diagonals). Click here to expand this figure. Figure EV1. Identification of 3,739 "oscillating" genes A. Overview of time courses in this study. B. Pairwise correlation of log2-transformed count data (n = 19,934) of the early time course (TC1) with the long developmental time course (TC2). High correlation is detected for samples that correspond to the same time points, justifying a fusion of these time courses to one continuous full developmental time course (TC3). C. Smooth scatter of amplitude over lower boundary of 99% confidence interval of the amplitude as determined by cosine fitting and error propagation (see Materials and Methods, related to Fig 1B). D, E. Scatterplot (D) of the peak phase of the long developmental time course (TC2) described here over the previously published L3-YA time course (TC6) (Hendriks et al, 2014). Genes that were identified as "oscillating" in both time courses (n = 2,499) are shown. Peak phases correlate well as confirmed by the coefficient of determination, R2, as indicated. However, they differ systematically (E) because a peak phase of 0° is arbitrarily chosen. A red vertical line indicates the mean phase difference (TC2 – TC6; corrected for circularity as described in Materials and Methods). Note that the gene-specific peak phase calculated here and previously both also differ from the arbitrarily assigned cycle phases in Appendix Fig S7 and their discussion. Download figure Download PowerPoint Figure 1. A massive oscillator with dispersed peak phases in several tissues Pairwise correlation plot of log2-transformed expression patterns of all genes (n = 19,934) obtained from a synchronized population of L1 stage larvae sampled and sequenced from t = 1 h until t = 48 h (TC3; a fusion of the two time courses TC1 and TC2 after 13 h; Fig EV1A and B). An asterisk indicates an outlier, time point t = 40 h. Scatter plot identifying genes with oscillatory expression (henceforth termed oscillating genes, blue) based on amplitude and 99% confidence interval (99%-CI) of a cosine fitting of their expression quantified on TC2 (Materials and Methods). A lower CI-boundary ≥ 0, i.e., P ≤ 0.01, and a log2(amplitude) ≥ 0.5, which corresponds to a 2-fold change from peak to trough, were used as cut-offs. Genes below either cut-off were included in the "not oscillating" group (black). Figure EV1C shows gene distributions in a density scatter plot. Gene expression heatmap of oscillating genes as classified in Figs 1B and EV1C. Oscillating genes were sorted by peak phase, and mean expression per gene from t = 7 h to t = 36 h (when oscillations occur) was subtracted. n = 3,680 as not all genes from the long time course (TC2) were detected in the early time course (TC1). Gray horizontal bars indicate the individual oscillation cycles, C1 through C4 which start at TP6, TP14, TP20, and TP27, respectively, as later determined in Appendix Fig S7. Radar chart plotting amplitude (radial axis, in log2) over peak phase (circular axis, in degrees) as determined by cosine fitting in Fig 1B. Enrichment (red) or depletion (blue) of tissues detected among oscillating genes expressed tissue-specifically relative to all tissue-specific genes using annotations derived from Cao et al (2017). Significance was tested using one-sided binomial tests which resulted in P-values < 0.001 for all tissues. Density plot of the observed peak phases of tissue-specifically expressed oscillating genes for all enriched tissues. Download figure Download PowerPoint The larger dataset enabled us to improve on the previous identification of genes with oscillatory expression (Hendriks et al, 2014). Using cosine wave fitting, and an amplitude cut-off of log2(amplitude) ≥ 0.5 and P ≤ 0.01, we classified 3,739 genes (24% of total expressed genes) as "oscillating" (i.e., rhythmically expressed) from TC2 (Figs 1B and EV1C and Dataset EV1; Materials and Methods). We confirmed this classification using MetaCycle (Wu et al, 2016, 2019), an algorithm that is widely used to study rhythmic circadian gene expression. At an FDR < 0.05 and an amplitude cut-off of log2(amplitude) ≥ 0.5, MetaCycle identified a comparable number, and highly overlapping set, of oscillating genes with similar amplitudes (Appendix Fig S1A and B). It also confirmed a predominant 7-h period for these genes (Appendix Fig S1C). We conclude that cosine fitting works robustly to identify oscillating genes in our data. Relative to the previous result of 2,718 oscillating genes (18.9% of total expressed genes) in mRNA expression data of L3 and L4 animals (Hendriks et al, 2014), this adds 1,240 new genes and excludes 219 of the previously annotated oscillating genes. We consider this latter group to be most likely false positives from the earlier analysis, resulting from the fact that some genes behave substantially different during L4 compared to the preceding stages as shown below. Visual inspection of a gene expression heatmap of the fused time course (TC3; Fig 1C) revealed four cycles of gene expression for the oscillating genes, presumably reflecting progression through the four larval stages. Oscillations were absent during the first few hours of larval development as well as in adulthood, from ~37 h on, and both their onset and offset appeared to occur abruptly. We will analyze these and additional features of the system and their implications in more detail in the following sections. Oscillating genes are expressed in several tissues with dispersed peak phases An examination of the calculated peak phases confirmed the visual impression that individual transcripts peaked at a wide variety of time points, irrespective of expression amplitude (Fig 1D). In circadian rhythms, peak phase distributions are typically clustered into three or fewer groups when examined in a specific tissue (Koike et al, 2012; Korenčič et al, 2014). However, the identity of oscillating genes differs across cell types and tissues, and for those genes that oscillate in multiple tissues, phases can differ among tissues (Zhang et al, 2014). Hence, we wondered whether the broad peak phase distribution was a consequence of our analysis of RNA from whole animals, whereas individual tissues might exhibit a more defined phase distribution. To understand in which tissue oscillations occur, we utilized a previous annotation of tissue-specifically expressed genes (Cao et al, 2017). 1,298, and thus a substantial minority (~35%) of oscillating genes, fell in this category for seven different tissues. They were strongly (~2.5-fold) enriched in the hypodermis (epidermis) and pharynx, and more modestly (≤ 1.5-fold) in glia and intestine (Fig 1E). By contrast, oscillating genes were greatly depleted from body wall muscle, neurons, and gonad. Hence, oscillatory gene expression occurs indeed in multiple tissues. However, although peak phase distributions deviated for each tissue to some degree from that seen for all oscillating genes, they were still widely distributed for each individual tissue (Fig 1F). We conclude that a wide dispersion of peak phases appears to be an inherent oscillator feature rather than the result of a convoluted output of multiple, tissue-specific oscillators with distinct phase preferences. Oscillations initiate with a time lag in L1 The observation that oscillations were undetectable during the first few hours of larval development and started only after > 5 h into L1 (Fig 1A and C) surprised us. Hence, we performed a separate experiment that covered the first 24 h of larval development (TC4). This confirmed our initial finding of a lack of oscillations during the first few hours of larval development (Fig 2A and B). Figure 2. Oscillations start with a time lag in L1 Gene expression heatmap of detectably expressed oscillating genes sampled from a separate early developmental time course (TC4; t = 1 h to t = 24 h). Genes were ranked according to their peak phase determined in Fig 1. Pairwise correlation plot of log2-transformed oscillating gene expression data obtained from both early larval development time courses, TC1 and TC4. Gene expression traces of the representative genes F11E6.3, col-68, and col-46. Scatter plot of calculated oscillating gene peak phase (as determined in Fig 1) over the time of occurrence of the first expression peak in L1 larvae, observed in TC4. Peak detection was performed using a spline analysis. As visual inspection did not reveal peaks in the heatmap during the first 3 h, and as the first cycle ends at 13 h, we performed this analysis for t = 3 h to t = 13 h to reduce noise. Generally, peak phases determined from the full developmental time course (TC3) correlate with the time of the first peak in L1. Data information: All analyses for oscillating genes identified in Fig 1 with detectable expression (n = 3,739 in A, n = 3,680 in B). Download figure Download PowerPoint To understand how oscillations initiate after the initial quiescence, we looked at individual genes and observed that the start of detectable oscillations differed for individual genes (Fig 2A and C). Nonetheless, the occurrence of first peaks was globally well correlated with the peak phases calculated from data in Fig 1 (Fig 2D); i.e., the order in which the first peaks of gene expression occurred was the same as the order given by the peak phases calculated for the stably running oscillator. (The apparent discontinuity in the data at a first peak of ~10 h is explained by the circularity of the data, with 0° = 360°, and the arbitrary assignment of 0° in the peak phase calculation.) Moreover, the transcript levels of many genes with a late-occurring (11–13 h) first peak proceeded through a trough before reaching their first peak as exemplified in Fig 2C for F11E6.3. We conclude that oscillations exhibit a structure of phase-locked gene expressing patterns as soon as they become detectable. L1 larvae undergo an extended intermolt Although the gene expression oscillations occur in the context of larval development, functional connections have been lacking. However, genes encoding cuticular components were reported to be enriched among previously identified oscillating genes (Kim et al, 2013; Hendriks et al, 2014), and Gene Ontology (GO-) term analysis of the new extended set of oscillating genes confirms that the top 12 enriched terms all linked to cuticle formation and molting, or protease activity (Fig 3A, Dataset EV2). These findings, and the fact that molting is itself a rhythmic process, repeated at the end of each larval stage, suggest the possibility of a functional link between molting and gene expression oscillations. Figure 3. Oscillatory gene expression is coupled to molting A. GO-term enrichments for oscillating genes as classified in Fig 1C. P-values were calculated using Fisher's exact test. The top 15 enriched terms are displayed. B. GFP signal quantification for qua-1p::gfp::pest::h2b::unc-543′UTR expressing single animals (HW2523, n = 20) over larval development, starting from hatch (t = 0 h). Individual traces are colored in black during the intermolt and in red during the molt. The mean intensity (blue line) and standard deviation across population (shading) are indicated. C, D. Boxplots of molt, intermolt, and larval stage durations (C) and of larval stage durations and period times of oscillations (D) of single animals (HW2523) developing in microchambers (n = 20). In (D), L1 was excluded because of the time lag before oscillations manifest after hatching. E. Boxplot of phase at molt entry (start of lethargus) and molt exit (end of lethargus) separated by larval stages for single animals (HW2523) developing in microchambers (n = 20) F. Scatterplot comparing developmental duration until second molt entry (M2 entry) with time to reach an arbitrarily chosen, unwrapped GFP oscillation phase obtained from data in (B). The particular phase chosen was observed close to the end of L2. The Pearson correlation is indicated with "r". G. Schematic model of expected phase variation at molt entry (gray) and molt exit (blue) depending on the coupling status between oscillations and molting. Width of colored blur is proportionate to observed standard deviation, sdobs, with sdobs,uncoupled > sdobs,coupled. H. Barplots displaying the ratio of observed standard deviation over expected standard deviation for phase calling from GFP intensity oscillations as measured in B, at either molt entry or molt exit for the indicated reporters. The empty bars indicate the expected value in the case of uncoupled processes . For coupled processes, we would expect 1. The values for all reporters are below 1. A dashed line indicates parity. (See Materials and Methods) I. Schematic depiction of coordination between oscillatory gene expression and development. Data information: Boxplots in (C–E) extend from first to third quartile with a line at the median, outliers are indicated with a cross, and whiskers show 1.5*IQR. Download figure Download PowerPoint If such a link were true, we would predict that the initial period of quiescence in the early L1 stage be accompanied by a lengthened stage, and, specifically, an extended intermolt duration. Indeed, using a luciferase-based assay that reveals the period of behavioral quiescence, or lethargus, that is associated with the molt (Appendix Fig S2A and B), others had previously reported an extended L1 relative to other larval stages (Olmedo et al, 2015). However, they reported an extension of both molt and intermolt. As the previously used luciferase-expressing transgenic strains developed relatively slowly and with limited synchrony across animals, presumably due to their specific genetic make-up, we repeated the experiment with a newly generated strain that expressed luciferase from a single-copy integrated transgene and that developed with improved synchrony and speed (Fig EV2A and B, Appendix Fig S2E–G, Materials and Methods). Our results confirmed that L1 was greatly extended relative to the other larval stages (Fig EV2E). However, in contrast to the previous findings (Olmedo et al, 2015), but consistent with our hypothesis, the differences appeared largely attributable to an extended intermolt (Fig EV2D). The duration of the first molt (M1) was instead comparable to that of M2 and M3 (Fig EV2C). Click here to expand this figure. Figure EV2. Quantification of stage durations reveals extended intermolt 1, intermolt 4, and molt 4 A. Representative raw luminescence traces of individual animal (HW1939) grown at 20°C. As the egg-shell is impenetrable to luciferin, a sudden increase in luminescence at the beginning of the time course indicates hatch (pre-hatch in red). Abrupt drops and subsequent rises in luminescence specify molts (in green). B. Heatmap showing trend-corrected luminescence (Lum.) trace for one animal per horizontal line (n = 86). Hatch is set to t = 0 h, and traces are sorted by time of entry into first molt. Blue indicates low luminescence and corresponds to the molts. C–E. Quantification of the duration of each molt (C), intermolt (D), and larval stage (E) in hours for HW1939 animals (n = 86). Data information: Boxplots in (C–E) extend from first to third quartile with a line at the median, outliers are indicated with a dot, and whiskers show 1.5*IQR. Download figure Download PowerPoint Thus, an extended first intermolt coincides with the fact that no oscillator activity can be detected by RNA sequencing during the first 5 h of this larval stage. Moreover, because we performed the experiment by hatching embryos directly into food, we can conclude that the extended L1 stage is an inherent feature of C. elegans larval development, rather than a consequence of starvation-induced synchronization. Development is coupled to oscillatory gene expression The luciferase assay revealed that also the L4 stage took significantly longer than the two preceding stages, though not as long as L1 (Fig EV2E). In this case, both the fourth intermolt and the fourth molt were extended (Fig EV2C and D). As apparent from the gene expression heatmap, and quantified below, the oscillation period during L4 was also extended. Hence, grossly similar trends appeared to occur in larval stage durations and oscillation periods, determined by the luciferase assay and RNA sequencing, respectively. We considered this as further evidence for a coupling of the two processes. To test this hypothesis explicitly, we sought to quantify the synchrony of oscillatory gene expression and developmental progression in individual animals at the same time. To this end, we established a microchamber-based time-lapse microscopy assay by adapting a previous protocol (Turek et al, 2015). In this assay, animals are hatched and grown individually in small chambers where they can be tracked and imaged while moving freely, enabling their progression through molts. Using Mos1-mediated single-copy transgene integration (MosSCI) (Frøkjær-Jensen et al, 2012), we generated transgenic animals that expressed destabilized gfp from the promoter of qua-1, a highly expressed gene with a large mRNA level amplitude. Consistent with the RNA sequencing data, we detected oscillations of the reporter with four expression peaks (Fig 3B). Moreover, we observed similar rates of development as in the luciferase assays when we curated the molts (Fig 3C, Appendix Table S1, Materials and Methods). Using a Hilbert transform (Pikovsky et al, 2001) to quantify the instantaneous, i.e., time-varying, changes in period (see Appendix), we observed that the averaged reporter oscillation period times for each cycle were in good agreement with the stage durations, for all three larval stages, L2-L4, for which oscillations period lengths could be reliably determined (Fig 3D). Single animal imaging enabled us to ask when molts occurred relative to oscillatory gene expression, and we observed a very uniform behavior across animals (Fig 3B, red segments). To quantify this relationship, we determined the gene expression phases at molt entries and exits. We obtained highly similar values across worms within one larval stage (Fig 3E), and only a minor drift when comparing phases across larval stages. Accordingly, when we plotted the times required to reach a specific, arbitrarily chosen oscillation phase in L2 or L3 over the time required to reach M2 or M3, we observed high levels of correlation (r > 0.9 for each instance; Fig 3F, Appendix Fig S3). Two additional reporter transgenes, based on the promoters of dpy-9 and F11E6.3, which differ in peak expression phases from qua-1 and one another, yielded similar results (Appendix Fig S4). We considered two possible interpretations of the close correlation between developmental progression and oscillations: First, both oscillations and development could be under independent, but precise temporal control. I
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