daf-42 is an evolutionarily young gene essential for dauer development in Caenorhabditis elegans

Daisy S. Lim, Jun Kim, Wonjoo Kim, Nari Kim, Sang-Hee Lee, Daehan Lee, Junho Lee

Preprint posted on 24 April 2023

DAFining dauer – a novel secreted factor determines developmental trajectory in nematode.

Selected by Chee Kiang Ewe

Categories: developmental biology


Dauer diapause – a state of hypobiosis – is widely conserved in many nematodes and appears to have driven the evolution of parasitism. For over five decades, free-living C. elegans has provided a convenient model to study the molecular mechanism of dauer formation. Dauers exhibit a distinct morphology which includes a specialized thickened cuticle with alae. Their body is constricted, and they develop a buccal plug that seals the mouth – all to promote survival in harsh environments (Cassada & Russell, 1975; Fielenbach & Antebi, 2008).

In C. elegans, the dauer developmental decision occurs at the late L1 stage though the commitment to dauer formation does not occur until mid-late L2d stage (Figure 1). Dauer entry is influenced by environmental factors (food availability, temperature, and pheromone) and is regulated by multiple signaling pathways, including the conversed insulin signaling pathway, TGFβ, and others. Mutants that show a dysfunctional dauer/adult developmental switch can be broadly divided into two classes: dauer-constitutive (Daf-c) and dauer-defective (Daf-d). While Daf-c mutants always enter dauer diapause, Daf-d animals never form dauer even in the presence of environmental triggers (Fielenbach & Antebi, 2008).

In this preprint, Lim and colleagues report a novel regulator of dauer development – DAF-42. Unlike the “classical” Daf-c and Daf-d that regulate the dauer entry decision, DAF-42 promotes L2d-to-dauer transition. Interestingly, despite the essential role of DAF-42 in dauer development, it is poorly conserved outside of the Caenorhabditis genus, suggesting rapid evolution of the dauer gene regulatory network.

Figure 1: C. elegans life cycle. C. elegans first stage (L1) larvae grows into reproductive adults in ~3 days at 20OC. In unfavorable growth conditions, the L1 animal can develop to form dauer, which can live several months without food. The dauer can resume reproductive development when the environment recovers.

Major findings:

  • daf-42 is a novel gene required for dauer entry.

By combining a screen for C. elegans mutants defective in dauer formation with genetic mapping-by-sequencing, the authors found that mutants lacking daf-42 failed to develop into dauer and would die when grown under dauer-inducing conditions. Next, the authors introduced the daf-42 mutation into worms carrying daf-2(e1370), a temperature sensitive Daf-c mutation, and could show that daf-2(-); daf-42(-) double mutants arrest at the L2d stage, while daf-2(-) single mutants robustly developed into dauers at 25oC. daf-2(-); daf-42(-) double mutants showed a degraded pharynx and molting defects. Moreover, dauer alae and cuticles were not properly formed in daf-2(-); daf-42(-) animals, indicating that daf-2(-); daf-42(-) animals can initiate the dauer formation program but fail to complete the developmental transition. Supporting this notion, removing DAF-7 (TGFβ ligand) or DAF-9 (cytochrome p450 enzyme in the steroid hormone pathway), both of which regulate the dauer entry decision, failed to rescue daf-42(-) lethality as DAF-42 functions downstream of the decision point influenced by harsh environmental conditions.

  • DAF-42 is an unstructured secreted protein that promotes L2d-to-dauer transition.

The authors reported that DAF-42 is a large protein with 17 isoforms. It is largely disordered and contains a signal peptide at the N terminus.

By examining transcriptomic data and transgenic reporters, the authors found that daf-42 is expressed in the seam cells (specialized hypodermal cells) during dauer entry. Indeed, expressing daf-42 under the control of a seam cell promotor could partially rescue the lethality of daf-2(-); daf-42(-) double mutants. The authors could show that DAF-42 is secreted from the seam cell to the peripheral hypodermis and cuticle during L2d stage, perhaps to promote molting during the L2d-to-dauer transition.

Next, the authors examined the transcriptomic differences between daf-2(-) single and daf-2(-); daf-42(-) double mutants and found that daf-2(-); daf-42(-) worms showed significant changes in gene expression during L2d, but not at an earlier point, supporting the model in which DAF-42 acts after dauer commitment in L2d. Intriguingly, many genes upregulated in daf-2(-); daf-42(-) are related to immune system and defense response.

  • daf-42 is a newly evolved gene in the Caenorhabditis

It is clear that DAF-42 plays an essential role in dauer development. Surprisingly, however, DAF-42 homologs are not found in nematodes outside of the Caenorhabditis genus. Within Caenorhabditis, the DAF-42 protein sequences exhibit substantial variation. These results indicate that the gene regulatory network for dauer development is highly plastic and a young gene has quickly evolved to gain essential developmental functions.

What I like about this preprint:

This preprint identifies an unusual daf gene that promotes dauer development. The data presented is solid. Most surprisingly, daf-42 appears to be evolutionary young and to have quickly evolved to adopt an essential developmental role. This study has shed light on the evolution of the nematode dauer signalling pathway and provides an interesting paradigm to further study the evolutionary trajectory of young genes.

Questions for the authors:

  • For the translational reporter, is it possible that not all isoforms are tagged, and daf-42 expression is not fully captured?
  • Have you looked closer at the fate of DAF-42 in the target tissues? Does it, for example, get taken up by the hypodermal cells?
  • It appears that DAF-42 may play an important role in molting as evident by the mutant phenotype and daf-42 expression pattern. Did you observe changes in the expression of genes regulating molting in the mutant?


Cassada, R. C., & Russell, R. L. (1975). The dauerlarva, a post-embryonic developmental variant of the nematode Caenorhabditis elegans. Developmental Biology, 46(2), 326–342.

Fielenbach, N., & Antebi, A. (2008). C. elegans dauer formation and the molecular basis of plasticity. Genes & Development, 22(16), 2149–2165.


Tags: dauer, developmental plasticity, diapause, molting

Posted on: 11 May 2023


Read preprint (1 votes)

Author's response

Junho Lee shared

  1. For the translational reporter, is it possible that not all isoforms are tagged, and daf-42 expression is not fully captured?

We think that all isoforms are tagged, because we used the genomic DNA for translational reporter, and GFP was tagged at the end of the last amino acid, which is common in all isoforms. All the exons and introns were included in the transgene.

  1. Have you looked closer at the fate of DAF-42 in the target tissues? Does it, for example, get taken up by the hypodermal cells?

We sought to find the localization of DAF-42 after molt into dauer stage, but the signal was too faint, probably due to degradation or diffusion, and we were unable to identify DAF-42::GFP localization. As of now, we do not know the fate of DAF-42 in the target tissues after dauer development.

  1. It appears that DAF-42 may play an important role in molting as evident by the mutant phenotype and daf-42 expression pattern. Did you observe changes in the expression of genes regulating molting in the mutant?

We did not investigate this in our manuscript, but analysis of our RNA-seq data shows increase of col-19, cut-2, mlt-3, col-14 and decrease of cut-2, mup-4, daf-12, lrp-1 in daf-2(-); daf-42(-) compared to daf-2(-) during L2d-to-dauer molting. The list of molting genes are from (Lazetic and Fay 2017) and Wormbase.

Reference: Lazetic, V., and D. S. Fay, 2017 Molting in C. elegans. Worm 6: e1330246.

gene name description Fold change (Log2) p value
col-19 Collagen – specific for the last molt 2.9702 1.97E-22
nas-37 Metalloprotease; promotes apolysis -1.5777 1.57E-14
cut-2 Cuticlin; present in all developmental stages 3.25913 3.34E-14
mlt-3 collagen and cuticulin-based cuticle. Involved in ecdysis. 1.1244 3.41E-08
col-14 Late collagen 1.00049 0.00087045
mup-4 Transmembrane protein; apical HDLS component -1.4403 0.00180796
daf-12 Nuclear hormone receptor – dauer-specific -1.3028 0.00220206
lrp-1 Low-density lipoprotein receptor; epidermal sterol absorption -1.3541 0.0094428

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