Abstract
Summary
The nematode Caenorhabditis elegans has six distinct life stages, embryo, four larval stages (L1-L4) and adult that are defined by landmarks such as hatch, ecdysis, as well as
associated stage-specific programs. lts development is strongly influenced by the environment: in favorable conditions, C. elegans develops rapidly to adult, whereas in
unfavorable conditions the worm can enter an alternative, reversible third larval stage, the dauer diapause.
Metazoan development is articulated through positional and temporal patterning at all life stages. ln C. elegans, a regulatory pathway, called the heterochronicircuit, has been
identified that controls temporal patterning in diverse tissues. In this study, we focused on the temporal control of development of third and later larval stages.
The nuclear hormone receptor daf-l2 functions within this heterochronic pathway regulating L3 options of reproductive growth and dauer diapause. daf-12 null mutants are fully dauer defective, but exhibit only weak heterochronic phenotypes suggesting that overlapping functions could act in concert with daf-l2 to specify developmental age.
We screened for !af-12 parallel or redundant functions (dre's) in a genetic screen for enhancement of the daf-12 heterochronic gonadal migration phenotypes. This screen
yielded two loci termed dre-1 and dre-2. dre-1 plays a novel role in C. elegans developmental timing of gonadal and
extragonadal tissues. In the heterochronic epidermal seam cell circuit, it is part of the late timer regulating the larval to adult switch. In the seam cells, dre-7 promotes L4 fates and prevents precocious expression of adult fates. Hence, absence of dre-1 results in precocious adult development of seam cells. Genetic epistasis analysis revealed that dre-l functions between the microRNA /ef-7 and the transcription factor lin-29, the latest acting gene that
promotes adult development. The comparably weak precocious phenotype of dre-l mutants may reflect the activity of other regulators at this step. Indeed, the timing of the larval to adult switch is regulated by at least four genes (lin-41, lin-42, hbl-l and dre-1). We found that dre-1 acts in parallel to lin-41 and lin-42 and may act in parallel or in the same pathway as hbt-l.
The gonad undergoes stage-specific migratory events (S1-S4 and SA) that are expressed in strict temporal sequence. However, little is known about the temporal control of
gonadal development. We found that developmental timing of the gonad is regulated by certain heterochronic activities as well, but it is organized differently and independently from the seam cell circuit. We identified four heterochronic gene products that function together. in a simple model, DRE-1 together with DAF-12 specifies S3/S4 programs, LIN-29 specifies 54 and the microRNA lin-4 specifies SA programs.
We cloned dre-1 and found that it encodes an evolutionary conserved F-box protein.
As an F-box protein containing a zinc finger (N-recognin class of zinc fingers of the UBRI protein family), it is likely to be involved in protein degradation. Orthologs are found in Caenorhabditis briggsae, Drosophila melanogasfer, Raffus noruegicus and humans. DRE-1 orthologs are uncharacterized proteins with unknown biological functions. Hence, the physiological role of DRE-1 in temporal control may be evolutionarily conserved. Moreover, genetic data suggest that DRE-1 functions in an SCF (Skp1, Cull and F-box) E3 ligase complex containing SKR-1 (C. elegans Skpl homolog) and CUL-1. Notably, developmental timing is regulated by transcriptional and translational control mechanisms. Therefore, the finding that dre-1 encodes an F-box seemingly involved in protein degradation adds a new
dimension to the regulation of temporal development.
Remarkably, dre-1 also affects a different developmental timer, the molt cycle, which is a measure of elapsed stages or chronological age. lt could affect both heterochronic and
molt circuits independently or act at the intersection of the two timers. dre-l null mutants develop to the three-fold stage of embryogenesis, but are unable to hatch, and partial
reduction of dre-1 results in molting defects at all larval molts. Thus, DRE-1 function may be required for hatching and molting, and suggests that key regulators of these processes may be targets of DRE-1 mediated degradation.
dre-1 was expressed prominently in the nucleus and reduced in the cytoplasm. lt was expressed in phenotypically affected tissues such as epidermal seam cells and gonadal
Distal Tip Cells that lead gonadal outgrowth, suggesting it could act cell autonomously.
Expression was also seen in the nervous system, excretory cells, body and pharyngeal muscle, and vulva, as well as other tissues.
Since daf-12 mutants affect C. elegans longevity, we asked whether dre-7 influences life span as well. We found thatdre-1 influences life span in a manner similar to daf-12, but to a lesser extent.
The dre screen yielded a second gene, dre-2 that is represented by a single allele.
dre-2 may play a role in C. elegans developmental timing as well. On its own it exhibited a modestly penetrant and strong delay in gonad migration, but no seam cell phenotypes.
However, it enhanced the retarded epidermal and gonadal phenotypes of lhe daf-12 null mutant and strongly suppressed lin-42 precocious adult alae phenotypes. Hence, dre-2may
represent a new heterochronic gene with auxillary functions. Alternatively, dre-2 could simply delay development non-specifically. A future challenge will be to clarify whether dre-2 represents a true heterochronic gene.
